NOS1-derived NO mediates S-nitrosation of SOCS1 and prevents SOCS1-mediated proteasomal degradation of p65. (A) Immunoblot analysis of p65 and SOCS1 in WT, NOS1^−/−, NOS2^−/−, and NOS3^−/− BMDM treated with LPS (250 ng/ml) for the indicated times. (B) SOCS1 S-nitrosation was detected using the biotin switch method on protein from WT and NOS1^−/− BMDMs pretreated with MG132 (50 µM) for 1 h before treatment with LPS (250 ng/ml) for the indicated times, followed by immunoblotting for SOCS1. (C) NOS2^−/− and NOS3^−/− BMDMs analyzed by biotin switch method, as in B. (D) Co-immunoprecipitation of SOCS1 and p65 in WT and NOS1^−/− BMDMs, pretreated with MG132 (50 µM, 1 h) before LPS (250 ng/ml) for the indicated time intervals. (E) Immunoblot analysis of p65 and SOCS1 total protein levels in NOS1^−/− BMDM treated with DEANO (NO donor, 5 µM) and LPS (250 ng/ml). All blots shown are representative of at least two (C and D) or three (A, B, and E) independent experiments.