48 hours later RGS14 expression level was analyzed by immunoblot with anti-myc antibodies
48 hours later RGS14 expression level was analyzed by immunoblot with anti-myc antibodies. 1 of the main manuscript for data analysis parameters). Data was fit by applying a one-site binding model including exothermic reaction phases (unfavorable enthalpy changes) with favorable free energy changes. Analysis of the data indicates that total saturation of the binding site is not achieved. This is likely due to the high dissociation rate of the complex.(0.66 MB TIF) pone.0004884.s001.tif (640K) GUID:?06DF0AE9-479A-4191-9BC1-C57B3E93A9B4 Physique S2: RGS14 selectively interacts with H-Ras and not other small GTPases in cells. HEK293T cells were transfected with plasmids encoding full-length myc-RGS14 and various mutationally-activated (GV, GD, or QL), HA-epitope tagged GTPases. Cell lysates were immunoprecipitated (IP) with anti-myc antibodies. Total lysates and precipitates were immunoblotted (IB) with indicated antibodies. Arf GTPase denotes the use of Arf1.(3.18 MB TIF) pone.0004884.s002.tif (3.0M) GUID:?720EABF5-EE9A-48EA-97EF-700341ECD679 Figure S3: Specificity of fluorescence complementation between H-Ras(G12S) and RGS14. HEK293T cells were co-transfected with cDNAs encoding the vacant vector pcDNA3.1, the N-terminal (amino acids 1C159) and C-terminal (amino acids 159-239) fragments of Yellow Fluorescent Protein (YFPN and YFPC), and indicated proteins fused to YFPN and YFPC. 48 hours after transfection, cells were analyzed by epifluorescence microscopy, and fluorescence was quantified using image analysis as explained in the Experimental section. (A) Transfection of the YFPC vector or YFPC-fusion constructs does not result in measurable fluorescence in the absence of YFPN co-transfection. (B) YFPN by itself does not go with YFPC nor YFPC-fusion constructs. (C) YFPN-H-Ras(G12S) suits both RGS14-YFPC and Raf-1-YFPC however, not YFPC nor YFPC-G1. (D) YFPN-G2 suits YFPC-G1 however, not YFPC, RGS14-YFPC, nor Raf-1-YFPC.(0.86 MB TIF) pone.0004884.s003.tif (844K) GUID:?F07B7963-E381-4221-8778-EE6437FA3CC8 Figure S4: Specificity of H-Ras/RGS14/B-Raf complex formation. HEK293T cells had been transfected with plasmids encoding full-length myc-RGS14, HA-B-Raf, and either G12V or wild-type HA-H-Ras. Cell lysates had been immunoprecipitated (IP) with anti-myc antibodies and proteins A/G agarose, as indicated. Total lysates and immunoprecipitates had been immunoblotted (IB) with indicated antibodies.(3.08 MB TIF) pone.0004884.s004.tif (2.9M) GUID:?2B381154-A922-4140-A045-43D06F0F10FE Body S5: Specificity and efficacy of rat RGS14 siRNAs (We). (A) HEK293T cells had been transfected with HA-epitope tagged RGS14 appearance vector and 6 hours afterwards transfected with control nonspecific (NS) siRNA or a pool of four RGS14 siRNAs. 24, 48, and 72 hours afterwards, RGS14 appearance level was examined by immunoblot (IB) with anti-HA. Examples had been immunoblotted with anti-actin antibodies being a control for total proteins amounts. (B) HEK293T cells had been transfected with myc-epitope tagged RGS14 appearance vector and 6 hours afterwards transfected with control nonspecific (NS) siRNA or four indie RGS14 siRNA duplexes (#1-4) that constitute the siRNA SMARTpool found in -panel A. 48 hours afterwards RGS14 appearance level was examined by immunoblot with anti-myc antibodies. Examples had been immunoblotted with anti-actin antibodies being a control for total proteins amounts.(1.89 MB TIF) pone.0004884.s005.tif (1.8M) GUID:?145089C4-434F-468C-A4FD-1087E3DF2092 Body S6: Specificity and efficacy of rat RGS12 and RGS14 siRNAs (II). Computer-12 cells had been transfected with control nonspecific (NS) siRNA, RGS12(D2) siRNA (duplex 2 from ref. 20), a SMARTpool (SP) of four RGS14 siRNA duplexes, or the four specific constituent siRNA duplexes (D1, D2, D3, and D4) which comprise the SMARTpool. 48 hours afterwards, cells had been gathered, RNA was extracted, and RGS12 (A) and RGS14 (B) appearance levels had been assessed by quantitative real-time PCR (as performed with the Gene Appearance Core from the UNC Dept. of Lab Pathology and Medication, aimed by Dr. Hyung-Suk Kim). RGS12 and RGS14 data had been normalized for comparative expression amounts using the 2-( delta delta Ct) technique with -actin as the inner control. Data is certainly presented as comparative expression in comparison to nonspecific (NS) siRNA treated examples. Statistical significance was motivated using ANOVA with Dunnett’s multiple evaluation check (* denotes P 0.5 vs NS siRNA samples).(0.30 MB TIF) pone.0004884.s006.tif (290K) GUID:?D3F3C2BA-CDD5-44C1-9599-7A8FDD19F14D Body S7: Yeast two-hybrid analysis of interactions between RGS14 and Ras-family GTPases. Fungus had been co-transformed with bait plasmids encoding indicated GTPase fusions using the Gal4p DNA binding area and victim plasmids encoding either Raf-1 or RGS14 fused towards the Gal4p activation area. Wild-type (WT) or glycine-12-to-valine (GV) mutationally-activated GTPases had been used to check for activation-dependent binding towards the Ras-binding area (RBD) of Raf-1 (proteins 50C131) as well as the tandem RBDs and GoLoco theme of RGS14 (proteins 263C544). Yeast had been plated on artificial described agar (SDA), missing leucine (-Leu, to choose for the pACT-II plasmid formulated with the Fusicoccin LEU2 gene), and tryptophan (-Trp, to choose for the pGBT9 Fusicoccin plasmid formulated with the TRP1 gene). Development on SDA-Leu-Trp demonstrates incorporation of bait and victim plasmids (best -panel). Development on SDA-Leu-Trp-His in the current presence of the histidine biosynthesis inhibitor 3-amino-1,2,4-triazole (3AT) signifies an optimistic protein-protein relationship.(2.04 MB TIF) pone.0004884.s007.tif (1.9M) GUID:?AC0C9794-E997-4ADA-9B35-C0D94869CC46 Desk S1: DNA constructs created and obtained for use in this research.(0.10 MB PDF) pone.0004884.s008.pdf (97K) GUID:?E1C302AF-8A30-4D12-BE10-A98A16F3AF19 Abstract Background Regulator of G-protein signaling (RGS) proteins have already been well-described as accelerators of G-mediated GTP hydrolysis (GTPase-accelerating proteins or GAPs). Nevertheless, RGS protein with complicated area architectures.20), a SMARTpool (SP) of four RGS14 siRNA duplexes, or the four person constituent siRNA duplexes (D1, D2, D3, and D4) which comprise the SMARTpool. one-site binding model concerning exothermic reaction stages (harmful enthalpy adjustments) with advantageous free energy adjustments. Analysis of the info indicates that full saturation from the binding site isn’t achieved. That is likely because of the high dissociation price of the complicated.(0.66 MB TIF) pone.0004884.s001.tif (640K) GUID:?06DF0AE9-479A-4191-9BC1-C57B3E93A9B4 Body S2: RGS14 selectively interacts with H-Ras rather than other little GTPases in cells. HEK293T cells had been transfected with plasmids encoding full-length myc-RGS14 and different mutationally-activated (GV, GD, or QL), HA-epitope tagged GTPases. Cell lysates had been immunoprecipitated (IP) with anti-myc antibodies. Total lysates and precipitates had been immunoblotted (IB) with indicated antibodies. Arf GTPase denotes the usage of Arf1.(3.18 MB TIF) pone.0004884.s002.tif (3.0M) GUID:?720EABF5-EE9A-48EA-97EF-700341ECD679 Figure S3: Specificity of fluorescence complementation between H-Ras(G12S) and RGS14. HEK293T cells had been co-transfected with cDNAs encoding the clear vector pcDNA3.1, the N-terminal (proteins 1C159) and C-terminal (proteins 159-239) fragments of Yellow Fluorescent Proteins (YFPN and YFPC), and indicated protein fused to YFPN and YFPC. 48 hours after transfection, cells had been examined by epifluorescence microscopy, and fluorescence was quantified using picture analysis as referred to in the Experimental section. (A) Transfection from the YFPC vector or YFPC-fusion constructs will not bring about measurable fluorescence in the lack of YFPN co-transfection. (B) YFPN by itself does not go with YFPC nor YFPC-fusion constructs. (C) YFPN-H-Ras(G12S) suits both RGS14-YFPC and Raf-1-YFPC however, not YFPC nor YFPC-G1. (D) YFPN-G2 suits YFPC-G1 however, not YFPC, RGS14-YFPC, nor Raf-1-YFPC.(0.86 MB TIF) pone.0004884.s003.tif (844K) GUID:?F07B7963-E381-4221-8778-EE6437FA3CC8 Figure S4: Specificity of H-Ras/RGS14/B-Raf complex formation. HEK293T cells had been transfected with plasmids encoding full-length myc-RGS14, HA-B-Raf, and either wild-type or G12V HA-H-Ras. Cell lysates had been immunoprecipitated (IP) with anti-myc antibodies and proteins A/G agarose, as indicated. Total lysates and immunoprecipitates had been immunoblotted (IB) with indicated antibodies.(3.08 MB TIF) pone.0004884.s004.tif (2.9M) GUID:?2B381154-A922-4140-A045-43D06F0F10FE Body S5: Specificity and efficacy of rat RGS14 siRNAs (We). (A) HEK293T cells had been transfected with HA-epitope tagged RGS14 appearance vector and 6 hours afterwards transfected with control nonspecific (NS) siRNA or a pool of four RGS14 siRNAs. 24, 48, and 72 hours afterwards, RGS14 appearance level was examined by immunoblot (IB) with anti-HA. Examples had been immunoblotted with anti-actin antibodies being a control for total proteins amounts. (B) HEK293T cells had been transfected with myc-epitope tagged RGS14 manifestation vector and 6 hours later on transfected with control nonspecific (NS) siRNA or four 3rd party RGS14 siRNA duplexes (#1-4) that constitute the siRNA SMARTpool found in -panel A. 48 hours later on RGS14 manifestation level was examined by immunoblot with anti-myc antibodies. Examples had been immunoblotted with anti-actin antibodies like a control for total proteins amounts.(1.89 MB TIF) pone.0004884.s005.tif (1.8M) GUID:?145089C4-434F-468C-A4FD-1087E3DF2092 Shape S6: Specificity and efficacy of rat RGS12 and RGS14 siRNAs (II). Personal computer-12 cells had been transfected with control nonspecific (NS) siRNA, RGS12(D2) siRNA (duplex 2 from ref. 20), a SMARTpool (SP) of four RGS14 siRNA duplexes, or the four specific constituent siRNA duplexes (D1, D2, D3, and D4) which comprise the SMARTpool. 48 hours later on, cells had been gathered, RNA was extracted, and RGS12 (A) and RGS14 (B) manifestation levels had been assessed by quantitative real-time PCR (as performed from the Gene Manifestation Core from the UNC Dept. of Lab Medication and Pathology, aimed by Dr. Hyung-Suk Kim). RGS12 and RGS14 data had been normalized for comparative expression amounts using the 2-( delta delta Ct) technique with -actin as the inner control. Data can be presented as comparative expression in comparison to nonspecific (NS) siRNA treated examples. Statistical significance was established using ANOVA with Dunnett’s multiple assessment check (* denotes P 0.5 vs NS siRNA samples).(0.30 MB TIF) pone.0004884.s006.tif (290K) GUID:?D3F3C2BA-CDD5-44C1-9599-7A8FDD19F14D Shape S7: Yeast two-hybrid.Seventy-two hours following transfection, neurite outgrowth was visualized by stage comparison microscopy and digital picture catch. binding site isn’t achieved. That is likely because of the high dissociation price of the complicated.(0.66 MB TIF) pone.0004884.s001.tif (640K) GUID:?06DF0AE9-479A-4191-9BC1-C57B3E93A9B4 Shape S2: RGS14 selectively interacts with H-Ras rather than other little GTPases in cells. HEK293T cells had been transfected with plasmids encoding full-length myc-RGS14 and different mutationally-activated (GV, GD, or QL), HA-epitope tagged GTPases. Cell lysates had been immunoprecipitated (IP) with anti-myc antibodies. Total lysates and precipitates had been immunoblotted (IB) with indicated antibodies. Arf GTPase denotes the usage of Arf1.(3.18 MB TIF) pone.0004884.s002.tif (3.0M) GUID:?720EABF5-EE9A-48EA-97EF-700341ECD679 Figure S3: Specificity of fluorescence complementation between H-Ras(G12S) and RGS14. HEK293T cells had been co-transfected with cDNAs encoding the bare vector pcDNA3.1, the N-terminal (proteins 1C159) and C-terminal (proteins 159-239) fragments of Yellow Fluorescent Proteins (YFPN and YFPC), and indicated protein fused to YFPN and YFPC. 48 hours after transfection, cells had been examined by epifluorescence microscopy, and fluorescence was quantified using picture analysis as referred to in the Experimental section. (A) Transfection from the YFPC vector or YFPC-fusion constructs will not bring about measurable fluorescence in the lack of YFPN co-transfection. (B) YFPN only does not go with YFPC nor YFPC-fusion constructs. (C) YFPN-H-Ras(G12S) matches both RGS14-YFPC and Raf-1-YFPC however, not YFPC nor YFPC-G1. (D) YFPN-G2 matches YFPC-G1 however, not YFPC, RGS14-YFPC, nor Raf-1-YFPC.(0.86 MB TIF) pone.0004884.s003.tif (844K) GUID:?F07B7963-E381-4221-8778-EE6437FA3CC8 Figure S4: Specificity of H-Ras/RGS14/B-Raf complex formation. HEK293T cells had been transfected with plasmids encoding full-length myc-RGS14, HA-B-Raf, and either wild-type or G12V HA-H-Ras. Cell lysates had been immunoprecipitated (IP) with anti-myc antibodies and proteins A/G agarose, as indicated. Total lysates and immunoprecipitates had been immunoblotted (IB) with indicated antibodies.(3.08 MB TIF) pone.0004884.s004.tif (2.9M) GUID:?2B381154-A922-4140-A045-43D06F0F10FE Shape S5: Specificity and efficacy of rat RGS14 siRNAs (We). (A) HEK293T cells had been transfected with HA-epitope tagged RGS14 manifestation vector and 6 hours later on transfected with control nonspecific (NS) siRNA or a pool of four RGS14 siRNAs. 24, 48, and 72 hours later on, RGS14 manifestation level was examined by immunoblot (IB) with anti-HA. Examples had been immunoblotted with anti-actin antibodies like a control for total proteins amounts. (B) HEK293T cells had been transfected with myc-epitope tagged RGS14 manifestation Fusicoccin vector and 6 hours later on transfected with control nonspecific (NS) siRNA or four 3rd party RGS14 siRNA duplexes (#1-4) that constitute the siRNA SMARTpool found in -panel A. 48 hours later on RGS14 manifestation level was examined by immunoblot with anti-myc antibodies. Examples had been immunoblotted with anti-actin antibodies like a control for total proteins amounts.(1.89 MB TIF) pone.0004884.s005.tif (1.8M) GUID:?145089C4-434F-468C-A4FD-1087E3DF2092 Shape S6: Specificity and efficacy of rat RGS12 and RGS14 siRNAs (II). Personal computer-12 cells had been transfected with control nonspecific (NS) siRNA, RGS12(D2) siRNA (duplex 2 from ref. 20), a SMARTpool (SP) of four RGS14 siRNA duplexes, or the four specific constituent siRNA duplexes (D1, D2, D3, and D4) which comprise the SMARTpool. 48 hours later on, cells had been gathered, RNA was extracted, and RGS12 (A) and RGS14 (B) manifestation levels had been assessed by quantitative real-time PCR (as performed from the Gene Manifestation Core from the UNC Dept. of Lab Medication and Pathology, aimed by Dr. Hyung-Suk Kim). RGS12 and RGS14 data had been normalized for comparative expression amounts using the 2-( delta delta Ct) technique with -actin as the inner control. Data is normally presented as comparative expression in comparison to nonspecific (NS) siRNA treated examples. Statistical significance was driven using ANOVA with Dunnett’s multiple evaluation check (* denotes P 0.5 vs NS siRNA samples).(0.30 MB TIF) pone.0004884.s006.tif (290K) GUID:?D3F3C2BA-CDD5-44C1-9599-7A8FDD19F14D Amount S7: Yeast two-hybrid analysis of interactions between RGS14 and Ras-family GTPases. Fungus had been co-transformed with bait plasmids encoding indicated GTPase fusions using the Gal4p DNA binding domains and victim plasmids encoding either Raf-1 or RGS14 fused towards the Gal4p activation domains. Wild-type (WT) or glycine-12-to-valine (GV) mutationally-activated GTPases had been used to check for activation-dependent binding towards the Ras-binding domains (RBD) of Raf-1 (proteins 50C131) as well as the tandem RBDs and GoLoco theme of RGS14 (proteins 263C544). Yeast had been plated on artificial described agar (SDA), missing leucine (-Leu, to choose for the pACT-II plasmid filled with the LEU2 gene), and tryptophan (-Trp, to choose for the pGBT9 plasmid filled with the TRP1 gene). Development on SDA-Leu-Trp demonstrates incorporation of bait and victim plasmids (best -panel). Development on SDA-Leu-Trp-His in the current presence of the histidine biosynthesis inhibitor 3-amino-1,2,4-triazole (3AT) signifies an optimistic protein-protein connections.(2.04 MB TIF) pone.0004884.s007.tif (1.9M) GUID:?AC0C9794-E997-4ADA-9B35-C0D94869CC46 Desk S1: DNA constructs created and obtained for.It really is of remember that YFPC-RGS14 complemented YFPN-H-Ras(G12S) with better performance than did YFPC-Raf-1 (Amount 4 and Amount S3), and with comparable performance towards the constitutive heterodimer of YFPC-G1 and YFPN-G2 (Amount S3). Open in another window Figure 4 Full-length H-Ras and RGS14 interact in live cells.HEK293T cells were transfected using the indicated combinations of plasmids, encoding the N-terminal fragment of Yellowish Fluorescent Protein (YFP) fused towards the N-terminus of H-Ras(G12S), as well as the C-terminal fragment of YFP fused towards the C-terminal of full-length Raf-1 or full-length RGS14, respectively. stages (detrimental enthalpy adjustments) with advantageous free energy adjustments. Analysis of the info indicates that comprehensive saturation from the binding site isn’t achieved. That is likely because of the high dissociation price of the complicated.(0.66 MB TIF) pone.0004884.s001.tif (640K) GUID:?06DF0AE9-479A-4191-9BC1-C57B3E93A9B4 Amount S2: RGS14 selectively interacts with H-Ras rather than other little GTPases in cells. HEK293T cells had been transfected with plasmids encoding full-length myc-RGS14 and different mutationally-activated (GV, GD, or QL), HA-epitope tagged GTPases. Cell lysates had been immunoprecipitated (IP) with anti-myc antibodies. Total lysates and precipitates had been immunoblotted (IB) with indicated antibodies. Arf GTPase denotes the usage of Arf1.(3.18 MB TIF) pone.0004884.s002.tif (3.0M) GUID:?720EABF5-EE9A-48EA-97EF-700341ECD679 Figure S3: Specificity of fluorescence complementation between H-Ras(G12S) and RGS14. HEK293T cells had been co-transfected with cDNAs encoding the unfilled vector pcDNA3.1, the N-terminal (proteins 1C159) and C-terminal (proteins 159-239) fragments of Yellow Fluorescent Proteins (YFPN and YFPC), and indicated protein fused to YFPN and YFPC. 48 hours after transfection, cells had been examined by epifluorescence microscopy, and fluorescence was quantified using picture analysis as defined in the Experimental section. (A) Transfection from the YFPC vector or YFPC-fusion constructs will not bring about measurable fluorescence in the lack of YFPN co-transfection. (B) YFPN by itself does not supplement YFPC nor YFPC-fusion constructs. (C) YFPN-H-Ras(G12S) suits both RGS14-YFPC and Raf-1-YFPC however, not YFPC nor YFPC-G1. (D) YFPN-G2 suits YFPC-G1 however, not YFPC, RGS14-YFPC, nor Raf-1-YFPC.(0.86 MB TIF) pone.0004884.s003.tif (844K) GUID:?F07B7963-E381-4221-8778-EE6437FA3CC8 Figure S4: Specificity of H-Ras/RGS14/B-Raf complex formation. HEK293T cells had been transfected with plasmids encoding full-length myc-RGS14, HA-B-Raf, and either wild-type or G12V HA-H-Ras. Cell lysates had been immunoprecipitated (IP) with anti-myc antibodies and proteins A/G agarose, as indicated. Total lysates and immunoprecipitates had been immunoblotted (IB) with indicated antibodies.(3.08 MB TIF) pone.0004884.s004.tif (2.9M) GUID:?2B381154-A922-4140-A045-43D06F0F10FE Amount S5: Specificity and efficacy of rat RGS14 siRNAs (We). (A) HEK293T cells had been transfected with HA-epitope tagged RGS14 appearance vector and 6 hours afterwards transfected with control nonspecific (NS) siRNA or a pool of four RGS14 siRNAs. 24, 48, and 72 hours afterwards, RGS14 appearance level was examined by immunoblot (IB) with anti-HA. Examples had been immunoblotted with anti-actin antibodies being a control for total proteins amounts. (B) HEK293T cells had been transfected with myc-epitope tagged RGS14 appearance vector and 6 hours afterwards transfected with control nonspecific (NS) siRNA or four unbiased RGS14 siRNA duplexes (#1-4) that constitute the siRNA SMARTpool found in -panel A. 48 hours afterwards RGS14 appearance level was examined by immunoblot with anti-myc antibodies. Examples had been immunoblotted with anti-actin antibodies being a control for total proteins amounts.(1.89 MB TIF) pone.0004884.s005.tif (1.8M) GUID:?145089C4-434F-468C-A4FD-1087E3DF2092 Amount S6: Specificity and efficacy of rat RGS12 and RGS14 siRNAs (II). Computer-12 cells had been transfected with control nonspecific (NS) siRNA, RGS12(D2) siRNA (duplex 2 from ref. 20), a SMARTpool (SP) of four RGS14 siRNA duplexes, or the four specific constituent siRNA duplexes (D1, D2, D3, and D4) which comprise the SMARTpool. 48 hours afterwards, cells had been gathered, RNA was extracted, and RGS12 (A) and RGS14 (B) appearance levels had been assessed by quantitative real-time PCR (as performed with the Gene Appearance Core from the UNC Dept. of Lab Medication and Pathology, aimed by Dr. Hyung-Suk Kim). RGS12 and RGS14 data had been normalized for relative expression levels using the 2-( delta delta Ct) method with -actin as the internal control. Data is usually presented as relative expression compared to non-specific (NS) siRNA treated samples. Statistical significance was decided using ANOVA with Dunnett’s multiple comparison test (* denotes P 0.5 vs NS siRNA samples).(0.30 MB TIF) pone.0004884.s006.tif (290K) GUID:?D3F3C2BA-CDD5-44C1-9599-7A8FDD19F14D Physique S7: Yeast two-hybrid analysis of interactions between RGS14 and Ras-family GTPases. Yeast were co-transformed with bait plasmids encoding indicated GTPase fusions with the Gal4p DNA binding domain name and prey plasmids encoding either Raf-1 or RGS14 fused to the Gal4p activation domain name. Wild-type (WT) or glycine-12-to-valine (GV) mutationally-activated GTPases were used to test for activation-dependent binding to the Ras-binding domain name (RBD) of Raf-1 (amino acids 50C131) and the tandem RBDs and GoLoco motif of.The RGS domain name is a 120 amino-acid nine-alpha helical bundle [5], [6] that contacts G subunits and thereby dramatically accelerates GTPase activity [7], [8]. the complex.(0.66 MB TIF) pone.0004884.s001.tif (640K) GUID:?06DF0AE9-479A-4191-9BC1-C57B3E93A9B4 Physique S2: RGS14 selectively interacts with H-Ras and not other small GTPases in cells. HEK293T cells were transfected with plasmids encoding full-length myc-RGS14 and various mutationally-activated (GV, GD, or QL), HA-epitope tagged GTPases. Cell lysates were immunoprecipitated (IP) with anti-myc antibodies. Total lysates and precipitates were immunoblotted (IB) with indicated antibodies. Arf GTPase denotes the use of Arf1.(3.18 MB TIF) pone.0004884.s002.tif (3.0M) GUID:?720EABF5-EE9A-48EA-97EF-700341ECD679 Figure S3: Specificity of fluorescence complementation between H-Ras(G12S) and RGS14. HEK293T cells were co-transfected with cDNAs encoding the vacant vector pcDNA3.1, the N-terminal (amino acids 1C159) and C-terminal (amino acids 159-239) fragments of Yellow Fluorescent Protein (YFPN and YFPC), and indicated proteins fused to YFPN and YFPC. 48 hours after transfection, cells were analyzed by epifluorescence microscopy, and fluorescence was quantified using image analysis as described in the Experimental section. (A) Transfection of the YFPC vector or YFPC-fusion constructs does not result in measurable fluorescence in the absence of YFPN co-transfection. (B) YFPN alone does not complement YFPC nor YFPC-fusion constructs. (C) YFPN-H-Ras(G12S) complements both RGS14-YFPC and Raf-1-YFPC but not YFPC nor YFPC-G1. (D) YFPN-G2 complements YFPC-G1 but not YFPC, RGS14-YFPC, nor Raf-1-YFPC.(0.86 MB TIF) pone.0004884.s003.tif (844K) GUID:?F07B7963-E381-4221-8778-EE6437FA3CC8 Figure S4: Specificity of H-Ras/RGS14/B-Raf complex formation. HEK293T cells were transfected with plasmids encoding full-length myc-RGS14, HA-B-Raf, and either wild-type or G12V HA-H-Ras. Cell lysates were immunoprecipitated (IP) with anti-myc antibodies and protein A/G agarose, as indicated. Total lysates and immunoprecipitates were immunoblotted (IB) with indicated antibodies.(3.08 MB TIF) pone.0004884.s004.tif (2.9M) GUID:?2B381154-A922-4140-A045-43D06F0F10FE Physique S5: Specificity and efficacy of rat RGS14 siRNAs (I). (A) HEK293T cells were transfected with HA-epitope tagged RGS14 expression vector and then 6 hours later transfected with control non-specific (NS) siRNA or a LAMC2 pool of four RGS14 siRNAs. 24, 48, and 72 hours later, RGS14 expression level was analyzed by immunoblot (IB) with anti-HA. Samples were immunoblotted with anti-actin antibodies as a control for total protein levels. (B) HEK293T cells were transfected with myc-epitope tagged RGS14 expression vector and then 6 hours later transfected with control non-specific (NS) siRNA or four impartial RGS14 siRNA duplexes (#1-4) that constitute the siRNA SMARTpool used in panel A. 48 hours later RGS14 expression level was analyzed by immunoblot with anti-myc antibodies. Samples were immunoblotted with anti-actin antibodies as a control for total protein levels.(1.89 MB TIF) pone.0004884.s005.tif (1.8M) GUID:?145089C4-434F-468C-A4FD-1087E3DF2092 Physique S6: Specificity and efficacy of rat RGS12 and RGS14 siRNAs (II). PC-12 cells were transfected with control non-specific (NS) siRNA, RGS12(D2) siRNA (duplex 2 from ref. 20), a SMARTpool (SP) of four RGS14 siRNA duplexes, or the four individual constituent siRNA duplexes (D1, D2, D3, and D4) which comprise the SMARTpool. Forty eight hours later, cells were harvested, RNA was extracted, and RGS12 (A) and RGS14 (B) expression levels were measured by quantitative real-time PCR (as performed by the Gene Expression Core of the UNC Dept. of Laboratory Medicine and Pathology, directed by Dr. Hyung-Suk Kim). RGS12 and RGS14 data were normalized for relative expression levels using the 2-( delta delta Ct) method with -actin as the internal control. Data is presented as relative expression compared to non-specific (NS) siRNA treated samples. Statistical significance was determined using ANOVA with Dunnett’s multiple comparison test (* denotes P 0.5 vs NS siRNA samples).(0.30 MB TIF) pone.0004884.s006.tif (290K) GUID:?D3F3C2BA-CDD5-44C1-9599-7A8FDD19F14D Figure S7: Yeast two-hybrid analysis of interactions between RGS14 and Ras-family GTPases. Yeast were co-transformed with bait plasmids encoding indicated GTPase fusions with the Gal4p DNA binding domain and prey plasmids encoding either Raf-1 or RGS14 fused to the Gal4p activation domain. Wild-type (WT) or glycine-12-to-valine (GV) mutationally-activated GTPases were used to test for activation-dependent binding to the Ras-binding domain (RBD) of Raf-1 (amino acids 50C131) and the tandem RBDs and GoLoco motif of RGS14 (amino acids 263C544). Yeast were plated on synthetic defined agar (SDA), lacking.