Sc 136967

Protein A/G beads, anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, mouse monoclonal or rabbit polyclonal antibodies specific for CCN3 (SC-136967), Twist (SC-81417), N-cadherin (ab76057), E-cadherin (ab40772), p-FAK (SC-11765-R), FAK (SC-932), p-Akt (SC-16646-R), Akt (SC-5298), HIF-1α (ab1), and β-actin (SC-130656) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Abcam (Cambridge, UK). Recombinant human CCN3 was purchased from PeproTech (Rocky Hill, NJ, USA). All other chemicals were obtained from Sigma–Aldrich (St Louis, MO).

A total of 30 μg of total protein from each condition was resolved by SDS-PAGE in 12% polyacrylamide gels prepared as described by [73 (link)]. Gels were blotted onto PVDF membranes, which were incubated with primary antibody diluted in 5% BSA (anti-CAPN1 diluted 1:500 (#2556; Cell Signaling, Danvers, MA, USA), anti-TGFB diluted 1:100 (ab92486; Abcam, Cambridge, UK), anti-CCN3, anti-GDF15 and anti-MIF diluted 1:100 and anti-CD9 diluted 1:200 (sc-136967, sc-377195, sc-271631, sc-13118 respectively; Santa Cruz Biotechnology, Heidelberg, Germany) overnight at 4 °C, washed and incubated with horseradish peroxidase-conjugated anti-mouse (R&D Systems, Minneapolis, MN, USA) or anti-rabbit (Jackson ImmunoResearch, Cambridgeshire, UK) antibodies for 2 h at RT. Immunoreactive bands were detected by enhanced chemiluminescence ECL (Millipore^®) according to the manufacturer’s instructions, and images were recorded using a ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA). Protein loading control was performed with Ponceau S staining.