Ab227502

RIPA buffer containing 1% protease inhibitor was added to cells or tissues, which were subsequently lysed on ice for 30 mins. The lysed cells or tissues were then centrifuged for 20 mins, and the supernatants were collected. The total protein concentration in each supernatant was determined by the BCA method. Next, a 30 μg aliquot of protein from each sample was separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Millipore, Burlington, MA, USA), which were subsequently blocked with 5% skim milk for 1.5 hrs. Next, the membranes were washed and then incubated overnight at 4°C with the following primary antibodies (1∶1000, Abcam, Cambridge, UK): IRAK1 (Abcam, ab238), E-cadherin (Abcam, ab15148), N-cadherin (Abcam, ab18203), vimentin (Abcam, ab137321), IL-1β (Abcam, ab9722), NLRP3 (Abcam, ab214185), ASC (Abcam, ab227502), ERK1/2 (Abcam, ab17942), JNK1/2 (Abcam, ab112501), p-ERK1/2 (Abcam, ab223500), p-JNK1/2 (Abcam, ab4821), Caspase 3 (Abcam, ab32042), and GAPDH (Abcam, ab9485). The next day, the membranes were washed and then incubated with a 1∶5000 diluted secondary antibody (Abcam) for 2 hrs. The immunostained proteins were detected using an ECL substrate kit (Thermo Scientific, Waltham MA, USA), and results were displayed on a Bio-Rad Gel Doc/Chemi Doc Imaging System.