B23 interferes with the association of GAPDH and SIAH1. (A) Mutagenesis of B23 at C275 removes its binding affinity for SIAH1. (B) B23 overexpression disrupts the interaction between SIAH1 and GAPDH. PC12 cells were transfected with FLAG-SIAH1 with or without GFP-B23. Cell lysates were immunoprecipitated with anti-FLAG antibody and analyzed by Western blotting. The binding between B23 and SIAH1 was stronger than that between SIAH1 and GAPDH. (C) B23 WT, but not B23 C275S, binds to SIAH1 and GAPDH to prevent the interaction between SIAH1 and GAPDH. (D) Depletion of B23 augments SIAH1 and GAPDH binding in PC12 cells after GSNO exposure. Cell extracts were immunoprecipitated with anti-SIAH1 antibody and analyzed by Western blotting. (E) Localization of B23, GAPDH, and SIAH1 in PC12 cells after B23 depletion and GSNO exposure. Cells were transfected with shB23 and FLAG-SIAH1 or MYC-GAPDH. After 48 h, cells were exposed to 200 µM GSNO for 18 h. FLAG or MYC tags were immunostained with anti–mouse–Alexa Fluor 594. B23 was immunostained with anti–rabbit–Alexa Fluor 488 and visualized with a confocal laser microscope (LSM 510). The nucleus was counterstained with Hoechst. IP, immunoprecipitation. Bars, 10 µm.