Trans blot turbo fast transfer system

Protein concentration was measured by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA, Cat No. 5000006). A total amount of 8 µg of protein was loaded per each lane of a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred to PVDF membranes using a Transblot Turbo fast transfer system (Bio-Rad). Membranes were blocked in 5% non-fat milk dissolved in Tris-buffered saline containing 0.05% Tween-20 (TBST). Primary antibody incubation was carried out at 4 °C overnight. Primary antibody dilutions were as follows: mouse anti-α-tubulin 1:4000 (Sigma-Aldrich, Cat No. T6199), rabbit anti-Histone H3 1:4000 (Abcam, Waltham, MA, USA, Cat No. 1791). After incubation with primary antibodies, membranes were washed in TBST three times and incubated with secondary antibodies for 1 h at room temperature. Secondary antibodies and their dilutions were as follows: goat anti-mouse 1:4000 (Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA, Cat No. 115-035-003), donkey anti-rabbit 1:2000 (Cytiva USA, Marlborough, MA, USA, Cat No. NA934). After secondary antibody incubations, membranes were washed in TBST again and developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Cat No. 34094). Bands were then visualized using a UVP Biospectrum AC imaging system.

Cells or islets were lysed in RIPA buffer containing 150 mM NaCl, 50 mM Tris, 1% v/v IGEPAL, 0.1% v/v SDS, and phenylmethylsulfonyl fluoride (PMSF) and subjected to sonication via a Bioruptor Plus (Diagenode, Denville, NJ). Extracts were run on Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) before being transferred to nitrocellulose membrane using a Trans-Blot Turbo fast transfer system (Bio-Rad). The membranes were blocked in 2.5% w/v BSA or 2.5% w/v milk in PBST before incubation with a primary antibody. Membranes were washed with PBST before incubation with HRP-conjugated or fluorescently-labeled secondary antibodies. The membranes were rewashed before imaging. Bands were detected with chemiluminescence (50 (link)) and visualized on ChemiDoc MP Imaging System (Bio-Rad). Antibodies used for Immunoblot detection are listed in Supplementary Table 2.