Hrp conjugated rabbit anti mouse secondary antibody

Live cells were lysed in protein extraction buffer [50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), 10 μM EDTA] with protease inhibitors for 30 min at 4°C. After a brief centrifugation, the supernatant of the cell lysate was collected and separated by 8% SDS–polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 2% non-fat milk in TBST (Tris-buffered saline; 100 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) followed by incubation with rabbit anti-LINE-1 ORF1p antibody (ab230966, Abcam) or rabbit anti-FAM208 (TASOR) antibody (ab224393, Abcam) at 4°C overnight. Membranes were washed three times with TBST and then incubated with a horseradish peroxidase (HRP)-conjugated mouse anti-rabbit secondary antibody (31464, Thermofisher Scientific) for 30 min at room temperature. After washing three times with TBST, proteins on the PVDF membrane were detected using an ECL reagent kit (P0018FS, Beyotime, Shanghai, China) and the ChemiScope 6000 Touch imaging system (Clinx, China). GAPDH was used as a loading control which was detected with mouse anti-GAPDH antibody (AF2819, Beyotime) followed by an HRP-conjugated rabbit anti-mouse secondary antibody (61–6520, Thermofisher Scientific).