Nupage 4 12 bistris mini protein gels

Cells were harvested and lysed in RIPA buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin (Cell Signaling Technology, Danvers, MA, USA) supplemented with both protease and phosphatase inhibitors (Roche Diagnostics, Rotkreuz, Switzerland). To ensure lysis, cells were sonicated with Diagenode Bioruptor Plus for 10 min (30 s on/30 s off cycle) at 4 °C and subsequently centrifuged at 4 °C at 13,000 rpm. Protein concentration was determined by the BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA) and an equal amount of protein was mixed with 4x NuPAGE LDS Sample Buffer (Invitrogen, Waltham, MA, USA) and 10× NuPAGE Reducing Agent (Invitrogen). Samples were heated at 70 °C for 10 min prior separation on NuPAGE 4–12% BisTris Mini Protein gels (Invitrogen) and NuPAGE MOPS SDS Running Buffer (Invitrogen). Proteins were subsequently transferred to polyvinylidene fluoride (PVDF) membrane via iBlot Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were incubated with the appropriate antibodies and imaged with Bio-rad ChemiDoc Imaging System using Western Lighting Plus ECL (PerkinElmer, Waltham, MA, USA). The list of antibodies and their sources can be found in Supplementary Table S2.