Sc 101494

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).

Twenty-five milligrams CD11b+ bone-marrow-derived myeloid cell or splenic T cell lysates were prepared as described for immunoprecipitation were electrophoresed on 10% SDS-PAGE gels and immunoblotted to detect MLCK210 (MYLK N-17, #sc-22223 or H195 Santa Cruz Biotechnology, #sc25428 each at 1:1000 dilution), integrin α4 (EPR1355Y, Abcam #ab81280 at 1:1000 dilution), HSP90 (Santa Cruz sc101494 at 1:1000 dilution), pSer18/pThr19 myosin light chain (Cell Signaling 3674 at 1:1000 dilution), myosin light chain 2 (Cell Signaling 3672 at 1:1000 dilution), pAkt (244F9, Cell Signaling at 1:1000 dilution), Akt (11E7, Cell Signaling at 1:1000 dilution), Flag tag (F7425, Sigma at 1:1000 dilution), myosin heavy chains A (Cell Signaling 3403 at 1:1000 dilution), myosin heavy chain B (Cell Signaling 3404 at 1:1000 dilution), myosin heavy chains C (Cell Signaling 3405 at 1:1000 dilution), paxillin (Santa Cruz 5574 at 1:1000 dilution), or Talin (Santa Cruz H-300 at 1:1000 dilution). All antibodies were diluted in TBST+ 5% BSA. Images of uncropped blots with molecular weight markers are included in the Source Data, available at 10.6084/m9.figshare.19358147.v1 and in the Source Data file that accompanies this article.