Ab101657

Total protein was extracted for western blotting analysis. The PVDF membranes were incubated overnight at 4°C with primary antibodies against COL6A6 (1:1000, PA5-60958, Thermo, USA), P4HA3 (1:1000, ab101657, Abcam, Cambridge, UK), E-cadherin (1:1000, ab1416, Abcam, Cambridge, UK), N-cadherin (1:1000, ab202030, Abcam, Cambridge, UK), Vimentin (1:1000, ab193555, Abcam, Cambridge, UK), p-PI3K(p85)(1:1000, #ab191606, Abcam, Cambridge, UK), PI3K(p85) (1:1000, #ab191606, Abcam, Cambridge, UK), and p-Akt (), Akt (), and subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (1:500, Abcam, Cambridge, UK). Signals were visualized using enhanced chemiluminescence reagent (Bio-Rad, USA).

The protein samples were acquired after cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was measured by a BCA kit (Beyotime), and the corresponding volume of protein (40 μg for each well) was added and mixed with loading buffer (Beyotime) in boiling-water bath for 3 min of denaturation. Electrophoresis was embarked for 30 min at 80 V and then for 1~2 h at 120 V once bromphenol blue reached the separation gel. After that, the proteins were transferred onto membranes at 300 mA for 60 min in ice-bath. Following rinsing 1~2 min with washing solution, the membranes were inactivated for 1 h at room temperature or sealed overnight at 4°C. The membranes were cultured with the primary antibodies against β-actin (ab8226, 1:1,000), Collagen I (ab34710, 1:1,000), α-SMA (ab32575, 1:1,000), P4HA3 (ab101657, 1:1,000), JNK (ab179461, 1:1,000), and p-JNK (ab124956, 1:1,000) (Abcam, Cambridge, MA, USA) at room temperature in a shaking table for 1 h. Before and after 1 h of incubation with the secondary antibody at room temperature, the membranes received 3 × 10 min of washing with washing solution. The chemiluminescence imaging analysis system (Gel Doc XR, Bio-rad) was applied for observation after membranes were given to developing liquid.