Anti hdac3 antibody

Cell lysates were loaded in a 10% polyacrylamide gel for electrophoresis, followed by nitrocellulose membrane transference based on the standard Western blot assay protocol. 5% non-fat milk in the PBS for blocking was incubated at 37 °C for 1 h. A corresponding antibody against a specific protein (anti-HDAC3 antibody was obtained from Proteintech, USA) was added for incubation at 4 °C overnight and followed by the detection with an appropriate horseradish peroxidase conjugated secondary antibody (1/1000, Cell Signaling Technology, USA) at 37 °C for 1 h, as described in our previous study [12 (link)]. After the final PBS washing, signal was developed by ECL detection system and relative photographic density was quantitated by a gel documentation and analysis system (Alpha Imager 2000, Alpha Innotech Corporation, USA).

Protein samples were separated by 8%–12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). After blocking with 5% milk in TBST for 1h, the membranes were incubated overnight at 4°C with primary antibodies. Subsequently, membranes were washed and incubated with secondary antibodies (1:10,000) and detected using the chemiluminescence method. The intensity of the bands was quantified by ImageJ software. Antibodies included anti-NLRP3 antibody (1:1,000, CST), anti-GSDMD antibody (1:1,000, Abcam), anti-N-GSDMD antibody (1:1,000, Abcam), anti-Capspase-1 p20 antibody (1:1,000, AdipoGen), anti-4-HNE antibody (1:1,000, Abcam), anti-HDAC3 antibody (1:1,000, Proteintech), anti-HADHA antibody (1:1,000, Abcam), anti-Ace-lys antibody (1:1,000, Abcam), anti-H3 antibody (1:1,000, Proteintech), anti-COX4 antibody (1:1,000, Abcam), anti-PCNA antibody (1:1,000, Abcam). Anti-GAPDH (1:5,000, Invitrogen) was used as an internal control.
For acetylation-immunoprecipitation, cells were lysed with immunoprecipitation buffer [supplemented with TSA (10 mM)] and sonicated. The samples were immunoprecipitated with protein A/G beads (Sigma) overnight at 4°C, washed three times in lysis buffer, resolved by loading buffer, and analyzed by Western blotting.