Horseradish peroxidase linked anti rabbit igg

At the end of the incubation periods, islets and INS-1E cells were washed in ice-cold PBS and lysed in RIPA lysis buffer containing 50 mM Tris HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS supplemented with Protease- and phosphatase inhibitors (Pierce, Rockford, IL, USA). Protein concentrations were determined with the BCA protein assay (Pierce). Equivalent amounts of protein from each treatment group were run on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and electrically transferred onto PVDF membranes. After blocking by 2.5% milk (Cell Signaling) and 2.5% BSA, membranes were incubated overnight at 4 °C with rabbit anti-cleaved caspase-3 (#9664), rabbit anti-PARP (#9532), rabbit anti-cleaved PARP (rat specific #9545), rabbit anti-phospho YAP(S127) (#4911), rabbit anti-LATS2 (#5888), rabbit anti-tubulin (#2146), rabbit anti-GAPDH (#2118), rabbit anti-β-actin (#4967) (all Cell Signaling Technology), and rabbit anti-PDX1 (#47267) and rabbit anti-p-MST1 (#79199) (both from Abcam) antibodies, all at a dilution of 1:1000, followed by horseradish-peroxidase-linked anti-rabbit IgG (Jackson). Membrane was developed by using a chemiluminescence assay system (Pierce) and analyzed using DocIT^®LS image acquisition 6.6a (UVP BioImaging Systems, Upland, CA, USA). Uncropped and unprocessed scans of all Western blots are available in the Source Data file.

Cells were washed with PBS, lysed in lysis buffer (see above) and protein concentration determined with the BCA protein assay (Pierce). In all conditions, the same amount of protein was run on a NuPAGE 4-12% Bis-Tris gel (Invitrogen) and electrically transferred onto PVDF membranes. After 1h blocking at RT, PVDF membranes were incubated with rabbit anti-Cleaved Caspase 3, rabbit anti-tubulin or rabbit anti-GAPDH (all Cell Signaling Technology) overnight at 4°C, followed by horseradish-peroxidase-linked anti-rabbit IgG (Jackson). PVDF Membranes was developed by chemiluminescence assay system (Pierce) and analyzed by DocIT®LS image acquisition 6.6a (UVP BioImaging Systems, Upland, CA, USA). For +control, INS-1E cells exposed to 12h palmitic acid were used as described before [61 (link)].

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).