Sk 0001

Following incubation, cells were washed with PBS three times and lysed in 250-μL lysis buffer (5-mM bicine buffer, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF 0.3 mM), leupeptin (10 μg/mL), and aprotonin (2 μg/mL)). Supernatants were obtained after centrifuging (8000 rpm) the cell lysates. Nuclear proteins were extracted to detect Snail using an extraction kit (SK-0001; Signosis, Santa Clara, CA, USA). Cells were collected and rinsed with ice-cold PBS, incubated in working reagent I for 10 min, and then centrifuged (8000 rpm) for 5 min at 4 °C. The supernatant was then collected and stored. The pellet was incubated in working reagent II for 2 h and centrifuged (8000 rpm) for 5 min. The supernatant was then collected for Snail detection. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The total proteins from cell lysates (25 μg) and nuclear proteins (25 μg) were electrophoresed in a 12.5% SDS-PAGE gel. After protein transfer, the PVDF membranes were blocked with 5% dehydrated skim milk and probed with primary antibodies (anti-human Akt, E-cadherin, FAK, MMP-2, MMP-9, N-cadherin, PI3K, Snail, TIMP-1, TIMP-2, mTOR, uPA, p-PI3K, p-Akt, p-mTOR, and actin antibodies) at 4 °C for 16 h, followed by secondary antibodies for 2 h. ECL reagent was used to detect protein expressions.

BMDMs were treated with different cytokines. Nuclear proteins were isolated with a nuclear extraction kit (Signosis, SK-0001), and analyzed by using a 96-well plate transcription factor (TF) activation array (Signosis, FA1002) according to the manufacturer’s instruction. The activity of each transcriptional factor was normalized as the fold of TFIID’s activity.