Anti h3 antibody

Dulbecco's modified Eagle's medium (DMEM), phosphate buffered saline (PBS), penicillin/streptomycin, fetal bovine serum were purchased from Wisent (Montreal, Canada). Dithiothreitol (DTT) was purchased from Merck (Whitehouse Station, NJ). Iodoacetamide (IAA) was purchased from Sigma (St Louis, MO). Sequencing grade modified trypsin was purchased from Promega (Fitchburg, WI). The TMT labeling kit was purchased from Thermo-Pierce Biotechnology (Rockford, IL). Anti-VIM antibody, anti-CHMP2B antibody, anti-PDZK1 antibody, anti-VTI1A antibody, anti-Cdk1 antibody, anti-Cdc25C antibody, anti-Y15-p-Cdk1 antibody and anti-H3 antibody were from Proteintech (Wuhan, China). Anti-ALDH1A1 antibody, anti-β-actin antibody and anti-flag antibody were from Sigma (St Louis, MO). Anti-CD133 antibody was from Miltenyi Biotec (Bergisch Gladbach, Germany).

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.

Cells were lysed with NETN buffer on ice for 20 min and then centrifuged at 12,000 rpm for 20 min to extract proteins. Then, the proteins were separated by SDS-PAGE. The following antibodies were used: anti-CDKL1 antibody (Abcam, ab136129), anti-YBX1 antibody (Proteintech, 20339-1-AP), anti-GAPDH antibody (Proteintech, 60,004-I-Ig), anti-Flag-tag antibody (ABclonal, AE005), anti-Myc-tag antibody (ABclonal, AE010), anti-H3 antibody (Proteintech, 17168-1-AP), anti-α-tubulin antibody (Proteintech, 11224-1-AP), and anti-PD-L1 antibody (Proteintech, 17952-1-AP).