With SH2-Domain ProteinsMERTK Interactions with SH2-Domain ProteinsFigure 6. Src is expressed and interacts with MERTK in the RPE. (A) Src family kinase and Hprt transcripts amplified from mouse RPE/choroid by RT-PCR. (B) Ni2+-NTA pull downs of recombinant SRC and HCK GST-SH2 domains incubated with or without 6xHis-rMERTK571?99 and evaluated on SDS-gels. (C) Src and Hck immunoreactivity on western blots of rat RPE/choroid and RPE-J cell homogenates. (D) Ni2+-NTA pull downs with RPE/ choroid protein homogenates from RCS congenic and dystrophic rats incubated with or without 25331948 6xHis-rMERTK571?99, and evaluated by western analysis with antibodies recognizing Src and Hck. (E) Src localization on cryosections of mouse retina/RPE/choroid. Details as in Figure 1. doi:10.1371/journal.pone.0053964.gAlexaFluor 488 anti-rabbit IgG (1:500); AlexaFluor 555 antimouse IgG. Images were obtained by confocal fluorescence microscopy (Leica SP5). For immunohistochemistry with RPE-J cells, the cultures were challenged with isolated bovine rod OS [50], washed 3 times with PBS, and fixed with 4 paraformaldehyde for 30 min at room temperature. The cells were then processed 25331948 using the same methods as for retinal cross sections.target plates and peptides were separated by liquid chromatography. Phosphopeptide analysis of the separated peptides was performed using a 4700 MALDI TOF/TOF mass spectrometer (Applied Biosystems) with peptide mass analysis using UniProt by the University of Michigan Protein Core Facility.rSH2-domain Protein Expression and PurificationGST-tagged constructs in pGEX2T vectors encoding the phosphotyrosine-recognition sequences of SH2-domain proteins were previously generated from a library representing nearly the complete set of known SH2-domain proteins [51]. The constructs were transformed into BL21 DE3 Gold bacteria and large scale cultures were grown in Terrific Broth with glycerol plus ampicillin at 37uC to an OD600 of 0.8. Daprodustat Isopropyl b-D-1-thiogalactopyranoside (final concentration 0.1 mM) was added and the cells were incubated at 15uC overnight. The cells were pelleted and resuspended in phosphate buffered saline and lysed by French press. Glutathione-agarose beads were incubated with cleared lysates for 1 h at 4uC, washed with 10 volumes of PBS- 1 TritonX100, and buy Dorsomorphin (dihydrochloride) eluted with buffer containing 8 mM glutathione, 50 mM Tris-HCl, pH 9.5. Fractions were collected and analyzed on SDS gels, and fractions containing rSH2-domains were pooled, concentrated to 1 mL, and loaded on a Sephacryl S-200 HR column (2161 in). Fractions were collected at a rate of 0.5 mL/ min, analyzed on SDS gels, and those containing purified rSH2domains were pooled and concentrated.rMERTK Pull Downs of rSH2-domains and Native SH2domain ProteinsPurified GST-tagged-rSH2-domain proteins (10 mg) were incubated with 6xHis-rMERTK571?99 (10 mg) in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and 0.1 mM PMSF for 1 h at 4uC. 50 mL of Ni2+-NTA resin slurry was added and the incubation was continued for an additional 0.5 h. The beads were collected by brief centrifugation, washed three times in binding buffer including 50 mM imidazole, and eluted with binding buffer containing 500 mM imidazole. Negative controls omitted the 6xHis-tagged-rMERTK571?99. For native tissue, protein homogenates from RPE/choroid were obtained as described for western analysis. Ni2+-NTA pull downs of the protein homogenates with 6xHis-tagged-rMERTK571?99 (10 mg) were performed as described above.Cell Cu.With SH2-Domain ProteinsMERTK Interactions with SH2-Domain ProteinsFigure 6. Src is expressed and interacts with MERTK in the RPE. (A) Src family kinase and Hprt transcripts amplified from mouse RPE/choroid by RT-PCR. (B) Ni2+-NTA pull downs of recombinant SRC and HCK GST-SH2 domains incubated with or without 6xHis-rMERTK571?99 and evaluated on SDS-gels. (C) Src and Hck immunoreactivity on western blots of rat RPE/choroid and RPE-J cell homogenates. (D) Ni2+-NTA pull downs with RPE/ choroid protein homogenates from RCS congenic and dystrophic rats incubated with or without 25331948 6xHis-rMERTK571?99, and evaluated by western analysis with antibodies recognizing Src and Hck. (E) Src localization on cryosections of mouse retina/RPE/choroid. Details as in Figure 1. doi:10.1371/journal.pone.0053964.gAlexaFluor 488 anti-rabbit IgG (1:500); AlexaFluor 555 antimouse IgG. Images were obtained by confocal fluorescence microscopy (Leica SP5). For immunohistochemistry with RPE-J cells, the cultures were challenged with isolated bovine rod OS [50], washed 3 times with PBS, and fixed with 4 paraformaldehyde for 30 min at room temperature. The cells were then processed 25331948 using the same methods as for retinal cross sections.target plates and peptides were separated by liquid chromatography. Phosphopeptide analysis of the separated peptides was performed using a 4700 MALDI TOF/TOF mass spectrometer (Applied Biosystems) with peptide mass analysis using UniProt by the University of Michigan Protein Core Facility.rSH2-domain Protein Expression and PurificationGST-tagged constructs in pGEX2T vectors encoding the phosphotyrosine-recognition sequences of SH2-domain proteins were previously generated from a library representing nearly the complete set of known SH2-domain proteins [51]. The constructs were transformed into BL21 DE3 Gold bacteria and large scale cultures were grown in Terrific Broth with glycerol plus ampicillin at 37uC to an OD600 of 0.8. Isopropyl b-D-1-thiogalactopyranoside (final concentration 0.1 mM) was added and the cells were incubated at 15uC overnight. The cells were pelleted and resuspended in phosphate buffered saline and lysed by French press. Glutathione-agarose beads were incubated with cleared lysates for 1 h at 4uC, washed with 10 volumes of PBS- 1 TritonX100, and eluted with buffer containing 8 mM glutathione, 50 mM Tris-HCl, pH 9.5. Fractions were collected and analyzed on SDS gels, and fractions containing rSH2-domains were pooled, concentrated to 1 mL, and loaded on a Sephacryl S-200 HR column (2161 in). Fractions were collected at a rate of 0.5 mL/ min, analyzed on SDS gels, and those containing purified rSH2domains were pooled and concentrated.rMERTK Pull Downs of rSH2-domains and Native SH2domain ProteinsPurified GST-tagged-rSH2-domain proteins (10 mg) were incubated with 6xHis-rMERTK571?99 (10 mg) in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and 0.1 mM PMSF for 1 h at 4uC. 50 mL of Ni2+-NTA resin slurry was added and the incubation was continued for an additional 0.5 h. The beads were collected by brief centrifugation, washed three times in binding buffer including 50 mM imidazole, and eluted with binding buffer containing 500 mM imidazole. Negative controls omitted the 6xHis-tagged-rMERTK571?99. For native tissue, protein homogenates from RPE/choroid were obtained as described for western analysis. Ni2+-NTA pull downs of the protein homogenates with 6xHis-tagged-rMERTK571?99 (10 mg) were performed as described above.Cell Cu.
