Anti ybx1 antibody

Cells cultured in 10-cm plates of 95% confluency were lysed in co-immunoprecipitation lysis buffer (Beyotime), briefly sonicated and centrifuged for clarification. Proteins were quantified using a BCA assay kit, then incubated with either agarose beads (Santa Cruz Biotechnology) and the intended antibodies including anti-APE1 antibody (Santa Cruz Biotechnology), anti-YBX1 antibody (Cell Signaling Technology), anti-PLK1 antibody (Proteintech), and anti-PPP1R12A antibody (Proteintech) or IgG (Proteintech) or with Anti-flag M2 beads (Sigma-Aldrich) at 4 °C overnight. Beads were washed four times with co-immunoprecipitation buffer with rotation at 4 °C for 5 min each time, boiled with 5× SDS loading buffer (Beyotime), clarified by centrifugation and loaded onto an SDS-PAGE gel for WB analysis as described above. For detecting interacting proteins in the cytoplasm, the cytoplasmic fraction obtained by subcellular fractionation was used.

ChIP–qPCR was performed using a MAGnity ChIP system (Invitrogen) according to the manufacturer’s protocol. In brief, cells were crosslinked and chromatin was extracted and sheared. Samples were immunoprecipitated with anti-5hMC antibody (Cell Signaling Technology, 51660S; clone HMC31) or anti-YBX1 antibody (Cell Signaling Technology, 9744; clone D2A11). The primer sequences used for ChIP–qPCR are as follows: GSDME, 5′-GACCCCTACTGCACTTCTGCAC-3′ (sense) and 5′-TGGGAAGAGACAGAGCCAAGAT-3′ (antisense); MUC1, 5′- CGGCCTGGGATA GCTTCCTC-3′ (sense) and 5′-TGCACTCCAGCCTGGGCG-3′ (antisense); and MUC13, 5′- GGAACTAGAGAGAGGGTGAGAAAGGGA-3′ (sense) and 5′-CTCTCTGATGGTCATGTCTAGCAAC-3′ (antisense). The results were from three independent experiments followed by normalization to input signals and shown as the mean ± s.d..