Las4000 lumi imager

Embryonic pancreases were lysed in triple detergent lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, 0.1% SDS, 1% NP40 and 0.5% Sodium Deoxycholate) and extraction of histones was optimized through the addition of HCl to a final concentration of 0.2 N. After 30 min incubation on ice, cell debris was pelleted and supernatants were quantified and prepared for SDS-PAGE electrophoresis on 16% Tris-tricine homemade gels. Proteins were then transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA) and incubated overnight at 4 °C with the antibodies indicated Table S3. Blots were visualized with ECL Reagent (Pierce Biotechnology, Rockford, IL, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY). Protein spots were quantitated with Image J software (http://rsb.info.nih.gov/ij/index.html).

For western blot analysis HUVECs were lysed in Radioimmunoprecipitation assay buffer (Sigma-Aldrich) adding 10 % proteases and 1 % phosphatases inhibitors (Sigma-Aldrich Química, S.L., Madrid, Spain). Protein content was determined using Bradford assay buffer (Sigma-Aldrich Química, S.L., Madrid, Spain). Fifty micrograms of lysates were separated by electrophoresis using polyacrylamide gel electrophoresis gels (4–12 %; LonzaIbérica S.A.U., Barcelona, Spain) and transferred to a polyscreen polyvinylidene difluoride membrane (Perkin Elmer, Waltham, MA, USA). After blocking with 5 % non-fat dried milk or 5 % bovine serum albumin, membranes were incubated with the respective primary antibodies—cleaved caspase3 (Asp175) antibody (#9661s) and p21 Waf1/Cip1 (12D1) antibody (#2947; Cell Signalling Technology)—overnight at 4 °C. The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated IgG antibodies (GE Healthcare Europe GmbH, Barcelona, Spain) at a 1:3,000 dilution for 1 h at room temperature. Blots were visualized with ECL reagent (Pierce Biotechnology, Rockford, IL, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY, USA). β-Actin—ACTB antibody (A-2066; Sigma-Aldrich Química, S.L., Madrid, Spain)—served as the loading control. Protein spots were quantitated with Image J software (http://rsb.info.nih.gov/ij/index.html).

Islets were lysed in triple detergent lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, 0.1% (w/v) SDS, 1% (v/v) NP40 and 0.5% (w/v) sodium deoxycholate). After 3 consecutive cycles of dry ice and 37 °C, cell debris was pelleted and supernatants were prepared for SDS-PAGE electrophoresis on 8% Tris-tricine homemade gels. Proteins were then transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA) and incubated overnight at 4 °C with the antibodies indicated in Supplementary Table 2. Immunoblots were developed with horseradish peroxidase-conjugated secondary antibodies (1:5000, GE Healthcare Bio-Sciences Corp. Piscataway, NJ, USA) and visualized with ECL Reagent (Pierce Biotechnology, Rockford, IL, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY). Protein spots were quantitated with Image J software. Due to the little amount of protein obtained from islets, protein extracts were not quantified but instead, the same number of islets was loaded in each lane. Representative images of the quantification results were selected. Uncropped blots are supplied in the Source Data file.