Iblot transfer stack to nitrocellulose

Frozen or fresh cell pellets (with 10⁶ cells) were denatured using 200ul SDS loading buffer (1x sodium dodecyl sulfate (Sigma-Aldrich), 1x protease inhibitor, 4 mM EDTA) and heated for 5mins at 68°C in a water bath. The heated cells were then centrifuged at 55,000 rpm for 1hr at 4°C. 30 ul of the supernatant was loaded onto a NuPAGE^™ 4–12% Bis-Tris Protein Gel (Thermo Fisher Scientific) and transferred using iBlot^™ Transfer Stack to nitrocellulose (Thermo Fisher Scientific) following the manufacturer’s protocol. The blot was then blocked in 1xTBST, 5% dry milk, 2% BSA for 1 h at room temperature, followed by overnight incubation at 4°C with the primary antibody, anti-SRSF1 antibody (ab133689) at 1:1000 and anti-GFP (ab1218) at 1:2000, together. The next day, the blot was washed with 1x TBST three times, then incubated with anti-rabbit and anti-mouse HRP conjugated secondary antibody (GE Healthcare Life Sciences) at 1:2000 for 2hrs at 4°C. The blot was then washed with 1x TBST at room temperature for 1hr and five more times for 3 min. Gel bands were detected using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and analyzed using a Bio-Rad ChemiDoc Image Lab 6.0 band analyzer.