Horseradish peroxidase hrp conjugated goat anti rabbit igg antibody

Cells were lysed in RIPA lysis buffer (Cell Signal Technology, Danvers, MA, USA) supplemented with protease inhibitors (Roche, Basel, Switzland) on ice. The lysates were collected and then subjected to ultrasonication and centrifugation at 9391g. for 15 min. The supernatants were collected, and protein content was determined by Bradford assay. Total proteins (30–50 μg) were separated by 10–12% SDS‐PAGE and blotted onto a nitrocellulose membranes (GE Healthcare, Marlborough, Massachusetts). The membrane was then blocked with 5% non‐fat milk in Tris‐buffered saline solution with Tween (TBS‐T) for 1 hr at room temperature and incubated with specific primary antibodies at 4°C overnight. After three 5‐min. washes in TBS‐T, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG antibody (1:2000; Abcam, Cambridge, UK) for 4 hrs. Then, the protein bands of interest were visualized with the Odyssey system (LI‐COR, Lincoln, Nebraska USA). The specific primary antibodies used were as follows: anti‐Slug (ab129153; Abcam), anti‐N‐Cadherin (#4061; CST, USA), anti‐E‐Cadherin (#3195; CST, USA), anti‐Vimentin (#5741; CST, USA) and anti‐β‐actin (#12620; CST, USA).

NSCLC lung tissues or cells were lysed on ice in RIPA lysis buffer (Beyotime, Shanghai) supplemented with protease inhibitors (Roche). The lysates were then collected and centrifuged at 14,000 rpm for 12 min. The supernatants were collected, and protein content was determined by Bradford assay. Total proteins were resolved using 12% SDS-PAGE separating gel (CWBiotech, Beijing, China) and blotted onto a PVDF membrane (Millipore, USA). Membranes were then blocked with 5% non-fat powdered milk intriethanolamine-buffered saline solution with Tween (TBS-T) at room temperature for 2 h, then incubated overnight with primary antibody at 4°C. Anti-p27(1:1000), Anti-p21(1:1000), anti-Bax (1:1000), anti-Cyclin D1 (1:1000), anti-Cyclin E (1:1000), anti-beta-actin (1:2000) or anti-Notch1 intracellular domain (NICD1) (1:2000). All primary antibodies were purchased from Abcam, USA. After three washes in TBS-T for 5 min, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:3000; Abcam) for 2 h at room temperature and washed three times in TBS-T, and visualized with an ECL Plus kit.