Cxcr4 and α sma

Whole cell lysates were prepared by scraping cells directly in 2x sodium
dodecyl sulfate (SDS) sample buffer. In parallel experiments, membrane proteins
were extracted using the Subcellular Protein Fractionation kit (Pierce
Biotechnology, Rockford, IL, USA) following the manufacturer’s
instructions. Extracellular matrix was prepared as we previously described
(4 (link)). Proteins were separated by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes.
Membranes were blocked with 5% non-fat dry milk in Tris buffered saline
(TBS)/0.05% Tween-20 (TBST) buffer then incubated with antibodies against human
IGFBP-4, Collagen, Fibronectin, Tenascin-C, CTGF, GAPDH (Santa Cruz, Dallas, TX,
USA), phosphorylated SMAD2 and SMAD3, total SMAD2 and SMAD3, phosphorylated
SMAD1/5/9, phosphorylated (p)-44/42 MAPK, p-AKT, p-P38 kinase, p-SAPK/JUNK and
antibodies targeting the corresponding total proteins (Cell Signaling Tech,
Danvers, MA, USA), CXCR4 and α-SMA (Abcam, Cambridge, MA, USA), His tag
(Sigma, St Louis, MO) or Tubulin (Epitomics Inc, Burlingame, CA, USA), washed
with TBS three times, then incubated with horseradish peroxidase-labeled
secondary antibody (Santa Cruz, Dallas, TX, USA). Signals were detected by
chemiluminescence (Perkin Elmer, Waltham, MA, USA) on a FluorChem R system
(ProteinSimple, San Jose, CA, USA). Images were analyzed using Image J.