Fig. 3

NOS1 induces S-nitrosylation of HDAC2-C262/274. a Control/NOS1 (A375) cells were treated with or without IFNα (1000 U/ml) for 6 h. Protein extracts were subjected to the biotin-switch assay. b Similar to a, but A375 cells were treated with or without L-NAME (1 mM), N-PLA (100 μM), 1400 W (100 μM) for 24 h. c Biotin-switch assay of A375 cells transfected with HDAC2-WT, HDAC2-C262A/C274A or empty vector (Control) for 24 h and then treated with GSNO (100 μM, 30 min). d Similar to c, but Control/NOS1 (A375) cells were used. e Control/NOS1(A375) cells were immunoprecipitated HDAC2 was subjected to HDAC activity assay. f Over-NOS1 (A375) cells were transfected with the indicated HDAC2 vectors. Flag antibody immunoprecipitates were subjected to HDAC assay. Shown are the averages and SEM (n = 3). g HDAC2 vectors were transfected into Over-NOS1 (A375) cells, followed by stimulation with IFNα (1000 U/ml) for 6 h and RT-PCR analysis. h HDAC2 vectors were transfected into Over-NOS1 (A375) cells and then treated with IFNα (1000 U/ml) for 6 h. HDAC2 immunoprecipitation was followed by ChIP-qPCR analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Flag-HD2, Flag-tagged HDAC2 expression vectors; End-HD2, endogenous HDAC2