Nitrocellulose transfer membranes

For western blot analysis, lysate proteins (45 μg) were separated by 12% or 15% w/v SDS-polyacrylamide gel electrophoresis. The separated proteins were then transferred onto nitrocellulose transfer membranes (0.2 or 0.45 μm, Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dry-milk in a buffer (10 mM Tris-HCL [pH 7.6], 100 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, incubated with the desired primary antibodies overnight at 4°C and then incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies at a 1 : 1000 dilution for 1 h at room temperature as previously described [27 (link)]. After washing, the proteins were detected using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Protein levels were quantified by densitometry using Quantity One software (Bio-Rad). Antibodies against β-actin, ERβ, Bad, and Bcl-xl were obtained from Cell Signaling Technology, and antibodies against Akt, p-Akt (Ser473), p-caspase 9, p-caspase 3, and cleaved PARP were obtained from Santa Cruz Biotech, Inc.

Proteins were extracted from HRECs or mouse retina tissues using radioimmunoprecipitation assay buffer (Sigma Aldrich, St. Louis, MI, USA) containing protease inhibitors and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Protein extracts (20 μg) were electrophoresed on a 12% (w/v) SDS-polyacrylamide gel and then transferred onto nitrocellulose transfer membranes (Millipore, Billerica, MA, USA) with pore size = 0.2 μm. Membranes were blocked with blocking buffer (Sigma Aldrich, St. Louis, MI, USA) and incubated with primary antibodies (Abs) overnight at 4 °C followed by incubation with horseradish peroxidase conjugated secondary antibody (GeneTex, TX, USA) at room temperature for 1 h. Primary Abs used in the study were RGC-32 (dilution 1:200, Santa Cruz Biotechnology, TX, USA), BCL2-associated X (Bax) (dilution 1:100, Thermo Scientific, Cheshire, UK), B-cell lymphoma 2 (Bcl-2) (dilution 1:1000, Cell Signaling Technology, Danvers, MA, USA), and cleaved caspase-3 (dilution 1:500, Cell Signaling Technology, MA, USA). Anti-β-actin (dilution 1:5000, Novus Biological, Centennial, CO, USA) was used as an internal control. Protein expression was detected with an enhanced chemiluminescence system (Syngene’s ChemiGenius XE Bio Imaging System, Frederick, MD, USA). Quantification analysis was performed using the ImageJ program and normalized to internal control.

Tissues, harvested from 6- to 9-week-old female C57BL/6 mice, were homogenized in cell lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100) containing complete protease inhibitors cocktail tablets (Roche Diagnostics). Isolated small intestinal epithelial cells, lamina propria lymphocytes (LPLs), and IELs were lysed in cell lysis buffer. Cell and tissue lysates were clarified by centrifugation, and protein concentration was measured with BCA Protein Assay Kit (Pierce). Ten micrograms of protein were denaturated in reducing sample buffer (NuPAGE LDS 4×; Novex^®, Life Technologies) containing 1M DTT (Sigma-Aldrich) and loaded onto a NuPage 4–12% Bis-Tris Gel (Novex^®, Life Technologies). Separated proteins were transferred onto nitrocellulose transfer membranes (Millipore) that were immunoblotted using anti-FLAG antibody (Sigma-Aldrich), anti-GFP antibody (Sigma-Aldrich), anti-Btnl6 rabbit polyclonal antiserum (Moravian-Biotech), rabbit preimmune serum, or anti β-actin antibody (Sigma-Aldrich), and detected with HRP-conjugated goat anti-mouse antibody or HRP-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch).