Anti pkcβii

The following antibodies were used: rabbit polyclonal antibodies against eIF6 [58 (link)], rpS6, phospho-rpS6 (Ser240/244), total 4EBP1 (Cell Signaling), P-PKCβII (Cell Signaling); anti-PKCβII (Santa Cruz); rabbit polyclonal anti-VEGFA (Abcam); mouse monoclonal antibodies against β-Actin (Sigma), PKCβ (BD-Bioscience) RACK1 IgM (BD Transduction Laboratories). Biotin was obtained from Pierce, EuroClone (EZ-LINK NHS-LC-BIOTIN). Lambda Protein Phosphatase (Lambda PP) was provided by NEB. Enzastaurin was provided by Eli Lilly and Company (Indianapolis, USA). eIF6 recombinant protein was produced in E. Coli by simultaneous co-expression with chaperones [21 (link)], followed by affinity chromatography and size exclusion chromatography (SEC; GE Healthcare).

The mitochondria (200 µg), obtained from CA2-4,DG hippocampi of gerbils subjected to 5 min ischemia and 1 h reperfusion, were lysed in 500 μL of lysis buffer: 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1mM PMSF. Samples were cleared of insoluble debris by centrifugation at 10,000× g for 20 min and preincubated with 20 µL protein A-Sepharose beads (Sigma-Aldrich, Poznan, Poland) to remove proteins nonspecifically associated with the beads. Resulting supernatant was incubated with anti-PKCβII (Santa Cruz Biotechnology, Dallas, TX, USA, Sc-13149)) or anti-GAC (Proteintech, Manchester, UK, 19958-1-AP) or anti-KGA (Proteintech, Manchester, UK, 20170-1-AP) antibodies conjugated to protein A beads according to the manufacturer protocol (Seize X Protein A Immunoprecipitation Kit, Pierce, Dallas, TX, USA). Proteins associated with antibodies were eluted with the electrophoresis sample buffer, resolved by 10% polyacrylamide gel electrophoresis and analyzed by reciprocal Western blots.