Bradford assay buffer

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).

PKCβ kinase activity was measured with a PKC Kinase Activity Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. HUVECs were cultured in NG, HG or HM conditions for 21 days, with or without Teneligliptin and/or GLP-1, and the medium was changed every 48 hours. At the end of incubation, the cells were lysed, the protein content was measured in Bradford assay buffer (Sigma-Aldrich Química, S.L., Madrid, Spain), and 30 μg of lysate was used to determine PKC-specific kinase activity. The assay was performed in triplicate, and the results are shown as arbitrary units (a.u.).