Western Blot Protein Analysis Protocol

Western blot analyses were performed as described previously [37 (link)]. The protein concentrations of cell or tissue lysates were measured using the Lowry assay. Afterward, 40 μg protein samples were separated using 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE, according to the molecular weight of the proteins of interest) and then electroblotted onto a nitrocellulose membrane. The membranes were blocked with BlockPRO blocking buffer (BP01-1L; Visual Protein, Taipei, Taiwan), immunoblotted with specific primary antibodies against THBS2 (1:1000, A8561; ABclonal, Woburn, MA, USA), HSP90 (1:500, sc-101494; Santa Cruz Biotechnology), transferrin (1:1000, GTX112729; Gene Tex, Irvine, CA, USA), CD 81 (1:500, GTX101766; Gene Tex), α-SMA (1:1000, bsm-52396R; Bioss Antibodies Inc, Woburn, MA, USA), β-catenin (1:1000, GTX632676; Gene Tex), Fibronetin (1:500, ab2413; Abcam, Cambridge, MA, USA), Vimentin (1:1000, SC-7557; Santa Cruz Biotechnology), and β-actin (1:1000, NB600-501; Novus Biologicals, Centennial, CO, USA), and then detected using horseradish peroxidase-conjugated secondary antibodies. The signals were visualized by chemiluminescence using an enhanced detection kit (ECL, TU-ECL03, TOOLS, Taipei, Taiwan).