Was subjected to immunoprecipitation using bead-conjugated anti-GFP. Total and immunoprecipitated proteins were subjected to Western analysis; equal Duvelisib amounts of different samples were loaded in each lane. Each candidate interactor was tested at least twice to confirm the immunoprecipitation result. The primary antibodies used were Rabbit anti-GFP (Abcam) and Mouse anti-V5 (Abcam).Split Ubiquitin Yeast Two-Hybrid AnalysesSplit-ubiquitin yeast two-hybrid assays were performed using the Dualsystems Biotech kit. Plasmids expressing TRPML1-CubLexA-VP16 and NubG or NubI-fusions were transformed into the yeast strain NMY51 [MATa his3delta200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4)] and selected on SD eu rp plates. Equal numbers of cells from each transformation were spotted on SD eu rp and eu rp ?ade is +1 mM 3-AT plates and incubated 23727046 at 30uC. Growth was scored over the next four days. For the yeast two-hybrid screens, the NMY51 yeast strain bearing a mouse TRPML1-Cub-LexA-VP16 expression plasmid was transformed with expression libraries for mouse cDNAs fused to NubG. The libraries used were a mouse heart X-NubG cDNA library (Dualsystems) and a mouse NubG-X cDNA library (generous gift of Igor Stagljar). Transformations were plated on SD eu rp plates to assess numbers screened and on SD eu rp de is +125 mM 3-AT plates to identify candidate interactors. More than 106 colonies were screened for each library. The NubG plasmid was isolated in Escherichia coli from each colony that grew on SD eu rp de is +125 mM 3-AT plates and was order GG918 retransformed into the NMY51 strain bearing an TRPML1-CubLexA-VP16 expressing plasmid to confirm the interaction. Once confirmed, each plasmid was sequenced to identify the cDNA/ gene and to confirm that the open reading frame was in-frame with NubG (those that were not in frame were discarded).GFP-TRPML1 Immunoprecipitation and Mass SpectrometryTo identify TRPML1-associated proteins, we immunoprecipitated GFP-TRPML1 (mouse) using bead-conjugated anti-GFP (MBL, Woburn, MA) from lysates of RAW264.7 macrophages stably expressing GFP-TRPML1 [19]. Lysis was done using Lysis Buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 NP40, 5 mM EDTA, 0.42 mg/ml sodium fluoride, 0.368 mg/ml sodium orthovanadate, 0.0121 mg/ml ammonium molybdate, 0.04 Complete protease inhibitors tablet/ml [Roche Diagnostics, Mannheim, Germany]) and washes were done using TNEN BufferGFP/TagRFP ImagingRAW264.7 macrophages stably expressing GFP-TRPML1 were transfected with plasmids expressing TagRFP(S158T) fusedProteins That Interact with TRPMLsample, and likewise, not all of the actual TRPML1 interactors may have been detected using this approach. We therefore decided to use a second technique, the Split-Ubiquitin Yeast TwoHybrid (SU-YTH) assay, to also screen for TRPML1 interactors. We reasoned that this complementary approach would generate a second list of candidates that we could compare to the Immunoprecipitation/Mass Spectrometry list to identify strong candidate TRPML1 interactors.Identification of TRPML1 Interactors by Split-Ubiquitin Yeast Two-Hybrid ScreensThe Split-Ubiquitin Yeast Two-Hybrid (SU-YTH) assay is a genetic method for in vivo detection of membrane-protein interactions that is based on the reconstitution of an ubiquitin molecule in Saccharomyces cerevisiae [30]. Because proteins are not targeted to the nucleus, this method allows for yeast two-hybrid analysis of full-length integral.Was subjected to immunoprecipitation using bead-conjugated anti-GFP. Total and immunoprecipitated proteins were subjected to Western analysis; equal amounts of different samples were loaded in each lane. Each candidate interactor was tested at least twice to confirm the immunoprecipitation result. The primary antibodies used were Rabbit anti-GFP (Abcam) and Mouse anti-V5 (Abcam).Split Ubiquitin Yeast Two-Hybrid AnalysesSplit-ubiquitin yeast two-hybrid assays were performed using the Dualsystems Biotech kit. Plasmids expressing TRPML1-CubLexA-VP16 and NubG or NubI-fusions were transformed into the yeast strain NMY51 [MATa his3delta200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4)] and selected on SD eu rp plates. Equal numbers of cells from each transformation were spotted on SD eu rp and eu rp ?ade is +1 mM 3-AT plates and incubated 23727046 at 30uC. Growth was scored over the next four days. For the yeast two-hybrid screens, the NMY51 yeast strain bearing a mouse TRPML1-Cub-LexA-VP16 expression plasmid was transformed with expression libraries for mouse cDNAs fused to NubG. The libraries used were a mouse heart X-NubG cDNA library (Dualsystems) and a mouse NubG-X cDNA library (generous gift of Igor Stagljar). Transformations were plated on SD eu rp plates to assess numbers screened and on SD eu rp de is +125 mM 3-AT plates to identify candidate interactors. More than 106 colonies were screened for each library. The NubG plasmid was isolated in Escherichia coli from each colony that grew on SD eu rp de is +125 mM 3-AT plates and was retransformed into the NMY51 strain bearing an TRPML1-CubLexA-VP16 expressing plasmid to confirm the interaction. Once confirmed, each plasmid was sequenced to identify the cDNA/ gene and to confirm that the open reading frame was in-frame with NubG (those that were not in frame were discarded).GFP-TRPML1 Immunoprecipitation and Mass SpectrometryTo identify TRPML1-associated proteins, we immunoprecipitated GFP-TRPML1 (mouse) using bead-conjugated anti-GFP (MBL, Woburn, MA) from lysates of RAW264.7 macrophages stably expressing GFP-TRPML1 [19]. Lysis was done using Lysis Buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 NP40, 5 mM EDTA, 0.42 mg/ml sodium fluoride, 0.368 mg/ml sodium orthovanadate, 0.0121 mg/ml ammonium molybdate, 0.04 Complete protease inhibitors tablet/ml [Roche Diagnostics, Mannheim, Germany]) and washes were done using TNEN BufferGFP/TagRFP ImagingRAW264.7 macrophages stably expressing GFP-TRPML1 were transfected with plasmids expressing TagRFP(S158T) fusedProteins That Interact with TRPMLsample, and likewise, not all of the actual TRPML1 interactors may have been detected using this approach. We therefore decided to use a second technique, the Split-Ubiquitin Yeast TwoHybrid (SU-YTH) assay, to also screen for TRPML1 interactors. We reasoned that this complementary approach would generate a second list of candidates that we could compare to the Immunoprecipitation/Mass Spectrometry list to identify strong candidate TRPML1 interactors.Identification of TRPML1 Interactors by Split-Ubiquitin Yeast Two-Hybrid ScreensThe Split-Ubiquitin Yeast Two-Hybrid (SU-YTH) assay is a genetic method for in vivo detection of membrane-protein interactions that is based on the reconstitution of an ubiquitin molecule in Saccharomyces cerevisiae [30]. Because proteins are not targeted to the nucleus, this method allows for yeast two-hybrid analysis of full-length integral.
