Anti flag antibody

Proteins samples separated by 15% SDS-PAGE and blotted to a PVDF membrane. Membranes were blocked for 1 h with 5% not-fat milk in TBST buffer, and probed 1 hour at 4°C either in TBST with a 1:2000 anti-Flag antibody (ZEN-BIOSCIENCE, Catalogue No. 700002), or with a 1:1000 anti-His tag antibody (ZEN-BIOSCIENCE, Catalogue No. 251784). After washing by TBST buffer three times, membranes were incubated for 1 h with HRP-conjugated secondary antibodies (1:10000, Goat Anti-Rabbit IgG H&L (HRP), ZEN-BIOSCIENCE, Catalogue No. 511203) in TBST buffer. Chemiluminescence substrate (Abbkine, Catalogue No. K22020) was added and signal quantifications were done in BioRad imager. As for the acetylation assay, the PVDF membranes were firstly probed by Acetylated Lysine Antibody (Affinity, Catalogue No. DF7729), then incubated with Goat Anti-Rabbit IgG (H + L) HRP (Affinity, Catalogue No S0001).

The cells were lysed on ice for 30 min in 1 mL lysis buffer (Servicebio, G2038), containing protease inhibitor MG132 (Selleck, S2619). After centrifugation at 17,000 g for 30 min, the supernatant was collected, where 100ul was used as the input, and the rest was shaken overnight at 4 °C with protein A + G Agarose IP reagent (Bioworld, BD0048) and 10 ul Anti-Flag antibody (ZENBIO, T201126-3A6). The beads were washed three times with lysis buffer and boiled in 2X SDS loading buffer for 5 min to release the bound proteins from the beads. Western blot was performed to detect the proteins in the IP samples.