Anti flag primary antibody

Cells in 24-well plates were washed carefully with ice-cold PBS buffer once, and then lysed with mammalian protein extraction buffer (GE) supplemented with HALT protease inhibitor cocktail (Fisher) with orbital shaking on ice for 20 min. The lysate was clarified by centrifugation at 21,000 rcf for 20 minutes at 4 °C. After boiling at 95 °C for 10 minutes in Laemmli buffer, the supernatants were run on 10% SDS-PAGE gels and transferred to a PVDF membrane (Millipore) following standard protocols. The membranes were blocked in 5% (w/v) non-fat milk (LabScientific) dissolved in TBS-T at room temperature for 1 hour. Anti-FLAG primary antibody (Proteintech, 20543–1-AP) and anti-GAPDH primary antibody (Proteintech, 10494–1-AP) were used at a 1:1,000 dilution in TBS-T and incubated at 4 °C overnight. The membranes were incubated in HRP-conjugated anti-rabbit IgG (CST, 7074S) at a 1:10,000 dilution in TBS-T for at room temperature for 1 hour. Membranes were then incubated with SuperSignal West Pico Chemiluminescent Substrate (Fisher) and visualized with a Bio-Rad ChemiDoc™ MP Imaging System. Quantification was performed by integrating the density of each band using ImageLab 5.0 software.