Western blotting was performed as depicted previously [3 (link)]. After the treatment periods, INS-1E cells or human islets were washed twice with ice-cold PBS and lysed with RIPA lysis buffer containing protease and phosphatase Inhibitors (Pierce, Rockford, IL, USA). Protein concentrations were measured by the BCA protein assay (Pierce). Lysates were fractionated by NuPAGE 4–12% Bis-Tris gel (Invitrogen) and electrically transferred onto PVDF membranes. Membranes were blocked in 2.5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) and 2.5% BSA (Sigma, St. Louis, MO, USA) for 1 h at room temperature and incubated overnight at 4 °C with rabbit anti-cleaved caspase-3 (#9664), rabbit anti-PARP (#9532), rabbit anti-cleaved PARP (rat specific #9545), mouse anti-Myc (#2276), rabbit anti-tubulin (#2146), rabbit anti-GAPDH (#2118) and rabbit anti-β-actin (#4967) (all Cell Signaling Technology), and rabbit anti-FBXO28 (#ab154068) (Abcam, Cambridge, UK) followed by horseradish-peroxidase-linked anti-rabbit IgG (Jackson, West Grove, PA, USA). Membranes were developed by a chemiluminescence assay system (Pierce) and evaluated with DocIT®LS image acquisition 6.6a (UVP Bio Imaging Systems, Upland, CA, USA).
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