Anti ha antibody 51 064 2 ap

The antibodies used for western blotting (WB), Co-IP, IHC and immunofluorescence (IF) in this study included antiSNAI2 antibody (9585, CST, USA), anti-USP7 antibody (66,514–1-lg, Proteintech, China), anti-TRIM21 antibody (12,108–1-AP, Proteintech, China), anti-CD34 antibody (11,265–1-AP, Proteintech, China), anti-Flag antibody (66,008–4-Ig, Proteintech, China), anti-Myc antibody (16,286–1-AP, Proteintech, China), anti-HA antibody (51,064–2-AP, Proteintech, China), mouse IgG (3420, CST),,protein A/G agarose beads, anti-Flag Magnetic Beads, anti-Myc Magnetic Beads, anti-HA Magnetic Beads (Beyotime, China) and MG132 (C2211, Sigma Aldrich) was dissolved in dimethyl sulfoxide.

Cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS) and lysed with RIPA buffer (Millipore) containing proteinase and phosphatase inhibitor cocktail (Roche) on ice for 30 min. The cell lysates (500–1000 μg) were incubated with 5 μg anti-HA antibody (51064–2-AP, Proteintech) and 50 μl protein G mag beads (GE Healthcare) on a rotating device at 4 °C overnight. Subsequently, pellets were washed three times with DPBS. Immunoprecipitated proteins were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (GE Healthcare). The membrane was blocked in 5% skim milk for 30 min and incubated with primary antibodies at 4 °C overnight. After being washed with TBST buffer, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 2 h. Peroxidase activity was detected using chemiluminescence by Luminata Forte Western HRP substrate (Millipore).