Amersham hybond ecl nitrocellulose membrane

Cellular subsets (3.5 × 10⁵ cells/subset) were resuspended in Laemmli sample buffer and heated in boiling water for 5 min. Total proteins were loaded on 10% Bis-Tris acryl-amide gels and blotted on AmershamTM HybondTM-ECL nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Rabbit anti-mouse GR (clone D6H2L) (Cell Signaling, MA, USA) and rabbit anti-mouse AKT (Cell signaling Technology, Danvers, MA) were used for protein detection. All primary antibodies were diluted in 5% BSA in PBST and blots were incubated overnight at 4°C.

Thymocytes (5 × 10⁶) were lysed in 25 μl RIPA buffer (25 mM Tris pH 8.0, 150 mM NaCl, 1.0% Triton-X, 0.1% SDS, 1% sodium deoxycholate, 1 mM EDTA) supplemented with complete protease inhibitors (Roche) and phosphatase inhibitors 1 mM Na₃VO₄ and 1 mM NaF. Proteins were separated on 12% SDS-polyacrylamide gels and electroblotted on AmershamTM HybondTM-ECL nitrocellulose membranes (GE Healthcare) according to standard protocols. Membranes were blocked in 5% (w/v) milk in PBST for 1 h and incubated with primary antibodies overnight. The following primary antibodies were used: rat anti-BIM (3C5/WEHI/Alexis), rabbit anti-BCL-xL (clone 54H6, Cell Signalling) and rabbit anti-ERK1/2 (clone 9102, Cell Signaling). All primary antibodies were diluted in 5% (w/v) milk in PBST. HRP-conjugated rabbit anti-rat IgG and goat anti-rabbit IgG (JacksonImmunoResearch Laboratories) were used as secondary antibodies. Immunoblots were developed using Western Bright ECL Spray (Advansta) and HRP chemiluminescence signals were detected with a Fujifilm LAS-4000 image analyzer (GE Healthcare) and analyzed with the Multi Gauge V3.0 software.

Total thymocytes as well as sorted DP and CD4SP thymocytes (2 × 10⁶) were lysed in 25 μl Carin Lysis buffer (20 mM Tris-HCl pH 8.0, 138 mM, NaCl, 10 mM EDTA, 1% Nonidet P-40, 10% glycerol) supplemented with complete protease inhibitors (Roche) and phosphatase inhibitors Na₃VO₄ (1 mM) and NaF (1 mM). Proteins were separated on 6% SDS-polyacrylamide gels and electroblotted on AmershamTM HybondTM-ECL nitrocellulose membranes (GE Healthcare) according to standard protocols. Membranes were blocked in 3% (w/v) milk in PBST for 1 h and incubated with primary antibodies for 3 hrs on room temperature. The following primary antibodies were used: rabbit polyclonal anti-NCOR1 (PA1-844A, Invitrogen), goat anti-NCOR1 (clone C-20, Santa Cruz) and mouse anti-α-Tubulin (clone DM1A, Sigma Aldrich). All primary antibodies were diluted in 3% (w/v) milk in PBST. HRP-conjugated goat anti-rabbit IgG and mouse anti-goat IgG (JacksonImmunoResearch Laboratories) were used as secondary antibodies. Immunoblots were developed using either Western Bright ECL Spray (Advansta) or Clarity Max Western ECL substrate (Bio Rad). HRP chemiluminescence signals were detected with a Fujifilm LAS-4000 image analyzer (GE Healthcare) and analyzed with the Multi Gauge V3.0 software.