Ab207299

Cells were washed twice with cold 1×PBS, and total lysates were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor cocktail (Pierce, Cramlington, UK) with mixing by sonication. The lysate concentration was measured using a bicinchoninic acid (BCA) standard curve. Lysates (40μg) were heated at 95°C for 5 min and separated by 10–12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After transferring onto polyvinylidene fluoride (PVDF) membranes and blocking with 5% milk, membranes were incubated with primary antibodies against PLCD1 (ab134936), MMP7 (ab207299; Abcam, Cambridge, MA, USA), KIF3A (#8507S), ERK1/2 (#4695), pERK1/2 (#4370), β-catenin (#8480), GSK-3β (#9315), pGSK-3β (#9323), β-actin (#4970; Cell Signaling Technology, Danvers, MA, USA), or active-β-catenin (05-665; Merck Millipore, Billerica, MA, USA) overnight, followed by secondary antibodies (1:5000 dilution, Cell Signaling Technology). Membranes were visualized using ECL Plus Detection Reagents (RPN2132, GE Healthcare Life Science, Amersham, UK).

The preparation of whole-cell extracts and western blot analysis were performed as previously described [4 (link)]. The proteins were determined using antibodies against MMP-7 (ab207299, Abcam, Cambridge, UK), MMP-10 (ab38930, Abcam), p-PI3K (#4228S, Cell Signaling Technology, Danvers, MA, USA), PI3K (#4292S, Cell Signaling Technology), p-AKT (#9271S, Cell Signaling Technology), AKT (#9272S, Cell Signaling Technology), p-mTOR (#5536S, Cell Signaling Technology), mTOR (#2972S, Cell Signaling Technology), IκBα (sc-371, Santa Cruz Biotechnology, Dallas, TX, USA), Snail (#3879, Cell Signaling Technology), and actin (sc-47778, Santa Cruz Biotechnology). After detection with the secondary antibodies, the proteins were determined using an enhanced chemiluminescence detection system. Actin served as a loading control. Densitometry data represent the mean ± standard error (SE) from three immunoblots and are shown as the relative density of protein bands normalized to the indicated protein (actin or total form of PI3K/AKT/mTOR).

Different groups of HCC cells were lysed on ice with RIPA buffer. Then, protein lysates were electrophoresed on 10% SDS polyacrylamide gels, transferred onto PVDF membranes (Millipore, USA), and blocked with 5% skim milk. Afterwards, primary antibodies: anti-RTKN2 (Abcam, #ab251807, 1:1000), anti-β-catenin (Abcam, #ab16051, 1:1000), anti-PCNA (Abcam, #ab29, 1:1000), anti-CyclinD1 (Abcam, #ab134175, 1:1000), anti-c-Myc (Abcam, # ab32072, 1:1000), anti-Bcl-2 (Abcam, # ab185002, 1:1000), anti-Bax (Abcam, # ab32503, 1:1000), anti-MMP-7 (Abcam, # ab207299, 1:1000), anti-E-cadherin (Abcam, #ab1416, 1:500), anti-N-cadherin (Abcam, #ab76057, 1:1000) were added at 4 °C overnight. Subsequently, secondary antibodies (1:1,000; Millipore, USA) was incubated for 1 h. Data was obtained and calculated using ImageJ software.