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. Author manuscript; available in PMC: 2022 Nov 7.
Published in final edited form as: Science. 2022 Aug 4;377(6606):621–629. doi: 10.1126/science.abm1638

Figure 4: KynA promotes GPR35-ATPIF1-ATP Synthase interaction.

Figure 4:

(A) Immunoblot analysis of anti-FLAG immunoprecipitates (FLAG IP) and whole cell extracts (WCE) from mouse neonatal GPR35−/− cardiomyocytes stably expressing GPR160-FLAG or GPR35-FLAG that were treated with 20 μM quinolinic acid, 20 μM KynA, 1 μM pamoic acid, 10 μM zaprinast or DMSO for 20 mins. (B) Immunoblot analysis of anti-FLAG immunoprecipitates from human AC16 cardiomyocytes stably expressing TMEM141-FLAG, GPR35 (wild-type)-FLAG or GPR35 (ΔC-terminus)-FLAG. (C) Immunoblot analysis of mitochondria isolated from mouse neonatal cardiomyocytes stably expressing GPR35-FLAG that were treated with 20 μM KynA for 20 mins. The isolated mitochrondria were incubated with increasing concentrations of proteinase K (PK) for 30 mins. (D) GPR35−/− mouse neonatal cardiomyocytes stably expressing FLAG-APEX-tagged wild-type or R151A GPR35 were treated with Biotin-Tryamide for 30 min and, where indicated, with 20 μM KynA for 20 min and H2O2 for 1 min (to enable biotinylation). Cell lysates (WCE) and biotinylated proteins captured on streptavidin agarose (STREP PD) were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against the indicated proteins or probed with horseradish peroxidase-conjugated streptavidin (Strep-HRP).