Goat anti rabbit 170 6515

Cells were harvested in ice-cold lysis buffer (150 mM NaCl, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.5) with added proteinase/phosphatase inhibitor (Roche). Cells were kept on ice with gentle agitation for 20 min to allow complete lysis. Lysate scraped into 1.5 ml tubes and cleared of debris by centrifugation at 14,000 × g for 20 min at 4°C. Supernatants were transferred to fresh tube and solubilized protein was measured using a DC protein assay kit (Bio-Rad). For immunoblotting, an appropriate volume of 1× Laemmli (Bio-Rad) sample loading buffer was added to the sample (10 µg of protein), which then heated at 90°C for 5 min before loading onto 4–20% gel (Bio-Rad). Proteins were separated using running buffer (Bio-Rad) for 2 hr at 150 V. Proteins were transferred to PVDF membrane (Bio-Rad) and membrane blocked in 5% (w/v) BSA in TBST or 5% (w/v) milk in TBST at RT for 2 hr. Blots were incubated with primary antibodies at 4°C overnight, followed by secondary antibody (Bio-Rad, Goat-anti-mouse #170–5047, Goat-anti-rabbit #170–6515, all used at 1:10,000) at RT for 1 hr. Membranes were washed three times and incubated in enhanced substrate Clarity (Bio-Rad) and imaged using a ChemiDoc XRS using Image Lab (Bio-Rad) for imaging and analyzing protein band intensities. ß-Actin or GAPDH levels were quantified to correct for protein loading.

Twenty-five to fifty micrograms of protein lysates were analyzed by SDS polyacrylamide gel electrophoresis (PAGE). After transfer to a 0.45-µm nitrocellulose membrane (GE Healthcare, Amersham, Thermo Fisher Scientific) and blocking in 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.05% (v/v) Tween 20 (TBST) containing 2.5% (w/v) skim milk, specific proteins were identified using primary antibodies for 16 to 18 h at 4 ℃. A secondary antibody [donkey anti-goat (sc-2020; Santa Cruz), goat anti-rabbit (1706515; Bio-Rad), or goat anti-mouse (1706516; Bio-Rad)] conjugated to horseradish peroxidase, and a luminol enhancer detection system (32106, Pierce, Thermo Fisher Scientific) were used to visualize the proteins.

Cells were washed with ice cold 1X PBS and lysed in ice-cold lysis buffer (150 mM NaCl, 20 mM HEPES, 1% NP-40, 5 mM EDTA, pH 7.5) with added proteinase/phosphatase inhibitor (Roche). The cell lysate was further sonicated (20% pulse frequency for 20 s) and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was collected and estimated for protein concentration using DC protein assay kit (Bio-Rad). For immunoblotting, an appropriate volume of 4 x Laemmli (Bio-rad) sample loading buffer was added to the sample (10–20 μg of protein), then heated at 90°C for 5 min before loading onto 4–20% gel (Bio-Rad). Proteins were separated using running buffer (Bio-Rad) for 2 hr at 110 V. Proteins were transferred to PVDF membrane (Bio-Rad) and membrane blocked in 5% (w/v) BSA or 5% (w/v) milk in TBST buffer (0.2 M Tris, 1.37 M NaCl, 0.2% Tween-20, pH 7.4) at room temperature for 1 hr. Blots were incubated with primary antibodies at 4°C overnight, followed by secondary antibody (Bio-Rad, Goat-anti-mouse #170–5047, Goat-anti-rabbit #170–6515, all used at 1:10,000) at room temperature for one hour. Membranes were washed three times and imaged by chemiluminescence (Pierce) by using a Chemidoc imaging system (BioRad). The images were further analyzed for band intensities using ImageJ software. β-Actin or GAPDH levels were quantified for equal protein loading.