Polyscreen polyvinylidene difluoride membrane

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).

Cells were lysed by using M-PER (Thermo Scientific) with additional protease inhibitors. Samples were separated by SDS-PAGE and transferred onto a PolyScreen polyvinylidene difluoride membrane (PerkinElmer). The membrane was immunoblotted with the indicated primary and appropriate secondary antibodies and detected using WesternBright ECL spray (Advansta). The images were captured using a BioSpectrum imaging system (UVP, Inc., LCC).