Anti pakt 1 2 3 ser 473 r

Cell lysates were prepared with the ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Burlington, Canada). Twenty µg of the cell lysate isolated from Rin-5F or islet cells or 20 µg proteins precipitated from the cell culture medium after cell treatment were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblot analysis was performed using anti-PARP-1 (H-250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PAR (H10; Alexis Biochemicals, San Diego, CA, USA), anti-Akt 1/2/3 (H-136; Santa Cruz Biotechnology), anti-pAkt 1/2/3 (Ser-473-R; Santa Cruz Biotechnology), anti-ERK 1/2 (K-23; Santa Cruz Biotechnology), anti-pERK (E-4; Santa Cruz Biotechnology) antibodies. Blots were probed with horseradish peroxidase-conjugated secondary antibodies. Staining was performed by the chemiluminescent technique according to the manufacturer's instructions (Amersham Pharmacia Biotech, Amersham, UK). If different set of samples were run on different gels, immunoblot analysis was performed in the same time, under the same conditions. All membranes were exposed under the same film and thus exposure and development did not influence the obtained results.

Immunoprecipitation was carried out with cell lysates (200 µg) obtained after lysis in 50 mM Tris 7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 1 mM PMSF, 1 µg/mL aprotinin, and 1 µg/mL leupeptin (lysis buffer). Cell lysates were incubated for 2 h on ice with 0.5 µg of anti-pAkt 1/2/3 (Ser-473-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pERK (E-4; Santa Cruz Biotechnology) antibodies or equal amount of control IgG (sc-2027; Santa Cruz Biotechnology). Protein A/G-coupled agarose beads (SC-2003; Santa Cruz Biotechnology) were added for 2 h at 4°C under constant agitation. The beads were pelleted and washed five times with lysis buffer. The immunoprecipitated proteins were resuspended in the sample buffer, separated by SDS-PAGE and analyzed by immunoblot analysis.

Cells were cultured in BD FalconTM 96-well black/clear bottom tissue culture plates optimized for imaging applications at 10,000 cells per well. After 24 h of incubation, cells were rinsed with PBS and fixed with 3.7 % formaldehyde solution at room temperature for 10 min. After fixation, cells were washed three times with PBS and permeabilized with 0.1 % Triton X-100 solution at room temperature for 5 min. Then, cells were washed twice with PBS, and non-specific binding was blocked by an addition of a 3 % FBS solution, and the cells were incubated at room temperature for 30 min. After that time, the cells were rinsed, incubated with rabbit polyclonal anti-p-EpoR [(Tyr 456)-R, Santa Cruz Biotechnology] and rabbit polyclonal anti-p-Akt1/2/3 [(Ser 473)-R, Santa Cruz Biotechnology] antibodies for 1 h at room temperature, washed three times with PBS and incubated with fluorescent (FITC) anti-rabbit secondary antibody for 60 min in the dark. After washing, nuclei were stained with Hoechst 33342 (2 lg/ml) and analyzed using confocal microscopy imaging.