Western lightning ultra detection kit

Total cellular proteins were extracted from cultured BMMs or OCs using RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate) supplemented with Protease Inhibitor Cocktail (Roche). Lysates were cleared by centrifugation at 16,000 g at 4°C for 20 mins and supernatants containing proteins were collected. For immunoblotting, 30μg of extracted proteins diluted in SDS-sampling buffer was resolved by SDS-PAGE (10–15%) gels and then electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham Life Science). Following transfer, membranes were blocked with 5% (w/v) skim milk in TBS-Tween (TBS; 0.05 M Tris, 0.15 M NaCl, pH 7.5 and 0.1% Tween-20) for 1 hr and then probed with primary antibodies diluted in 1% (w/v) skim milk powder in TBS-Tween at 4°C overnight. Membranes were washed and then incubated with HRP-conjugated secondary antibodies and antibody reactivity was detected by the Western Lightning Ultra Detection Kit (PerkinElmer, Waltham, MA, USA) using the FujiFilm LAS-3000 Gel Documentation System (FujiFilm, Tokyo, Japan) and its associated software.

Total cellular proteins were extracted from cultured cells using RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate) supplemented with Protease Inhibitor Cocktail (Roche). Lysates were cleared by centrifugation at 16,000 g at 4 °C for 20 mins and supernatants containing proteins were collected. For immunoblotting, 30 μg of extracted proteins diluted in SDS-sampling buffer was resolved by SDS-PAGE (10–15%) gels and then electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham Life Science). Following transfer, membranes were blocked with 5% (w/v) skim milk in TBS-Tween (TBS; 0.05 M Tris, 0.15 M NaCl, pH 7.5 and 0.1% Tween-20) for 1 h and then probed with primary antibodies diluted in 1% (w/v) skim milk powder in TBS-Tween at 4 °C overnight. Membranes were washed and then incubated with HRP-conjugated secondary antibodies and antibody reactivity was detected by the Western Lightning Ultra Detection Kit (PerkinElmer, Waltham, MA, USA) using the FujiFilm LAS-3000 Gel Documentation System (FujiFilm, Tokyo, Japan) and its associated software.

Total cellular proteins (TCP) were extracted using RIPA lysis buffer (150mM NaCl, 50mM Tris-HCl pH 8.0, 1.0% NP40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails (Sigma-Aldrich^®, USA) for 30mins on ice. Cellular debris were clarified by centrifugation at 16,000g for 20mins at 4°C. Cleared TCP were diluted with 4X SDS sampling buffer and boiled for 5mins. 30μg of TCP were separated on 10%-17.5% SDS-PAGE gel and resolved protein transferred to nitrocellulose membrane for analysis by western blot. Membranes were blocked with 5% skim milk in TBS-Tween (TBST) for 1hr and then incubate with primary antibodies diluted in 1% (w/v) skim milk in TBST for 2hrs shaking at RT or overnight shaking at 4°C. Membranes were washed three times with TBST and then incubated with the corresponding HRP-conjugated secondary antibodies. After three times TBST and three times TBS washes, antibody reactivity were detected using the Western Lightning Ultra Detection Kit (Perkin Elmer, USA) using the FujiFilm LAS-3000/4000 Gel Documentation System (Japan) and its associated software. Immunoblot images presented are representatives of at least three independent experiments.