Molecular imager chemidoc xrs with image lab software version 3

Protein extracts were prepared and analysed by western blots according to standard protocols as described previously [51 (link)] using primary antibodies, including ERK1/2 (1:2000, Cell Signaling Technology), phospho-p44/42 MAPK [ERK1/2(Thr202/Tyr204), 1:2000, Cell Signaling Technology], Akt (1:2000, Cell Signaling Technology), phosphor-Akt(Ser473) (1:2000, Cell Signaling Technology), and ANXA2 (1:2000, Cell Signaling Technology). Bands were detected using the Molecular Imager ChemiDoc XRS + with Image Lab Software version 3.0 (Bio-Rad, Hercules, CA, USA).

Whole-cell extracts were prepared using SDS lysis buffer (0.1 M Tris-HCl, 4% SDS, 0.2% bromophenol blue, and 5%  β-mercaptoethanol) with proteinase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined by a Micro BCA™ protein assay reagent kit (Pierce). After boiling for 10 min in sample buffer with sodium dodecyl sulfate and β-mercaptoethanol, the protein sample (7 μg) was electrophoresed through 10% acrylamide gels and transferred electrophoretically to nitrocellulose membranes (Amersham Biosciences). After blocking with 5% nonfat milk in wash buffer (20 mM Tris-HCl, 100 mM NaCl, 0.05% Tween20) for 2 hrs, the membranes were incubated at 4°C overnight with primary collagen I antibody (Col-I) at 1 : 2000 (Abcam), α-SMA antibody at 1 : 2000 (Abcam), AFP antibody at 1 : 2000 (R&D Systems), ALB antibody at 1 : 2000 (R&D System), CK19 antibody at 1 : 2000 (R&D Systems), or β-actin antibody at 1 : 6000 (Sigma-Aldrich). After three washes with wash buffer, the membranes were incubated for 1 hr with corresponding horseradish peroxidase-conjugated secondary antibody at 1 : 4000 (Sigma-Aldrich). Detection and quantification of bands were performed using the Molecular Imager Chemi Doc™ XRS+ with Image Lab Software version 3.0 (Bio-Rad).