Anti fak

The hBMSCs were lysed in NP-40 buffer on ice for 1 h and the suspensions were centrifuged at 14,000 g. The cell lysates were separated by SDS-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The membrane blocked in 3% bovine serum albumin (BSA) for 30 min and then immunoblotted with the primary anti-FAK, anti-phosphor-FAK, and β-actin (GeneTex, San Antonio, TX, USA) for 3 h, then washed three times by the tris-base saline buffer containing 0.05% Tween-20. A horseradish peroxidase (HRP)-conjugated secondary antibody was subsequently added and the proteins were visualized with enhancement using enhanced chemiluminescent detection kits (Invitrogen, Carlsbad, CA, USA). The stained band was scanned and quantified using a densitometer (Syngene bioimaging system, Frederick, MD, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA). The protein expression level was normalized to the β-actin for each group.

Primary antibodies against Fascin-1 [mouse monoclonal (D-10) sc-46675], RhoA [mouse monoclonal (26C4) sc-418], GAPDH [rabbit polyclonal (FL-335) sc-25778], Actin [goat polyclonal (C-11) sc-1615], GLUT1 [mouse monoclonal (A-4) sc-377228], GLUT4 [rabbit polyclonal (H-61) sc-7938] and SDF-1/CXCL12 [rabbit polyclonal (FL-93) sc-28876] were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-phospho-p44/42 ERK (Thr202/Tyr204) [mouse monoclonal, #9106S] and p44/42 ERK1/2 [rabbit polyclonal, #9102] from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-FAK [rabbit polyclonal, GTX132141] was from GeneTex, Inc. (Irvine, CA, USA); anti-MEK1[rabbit monoclonal, #04-376] was from EMD-Millipore (Burlington, MA, USA); anti-α-smooth muscle Actin (mouse monoclonal (1A4), #A2547) was from Sigma-Aldrich (Saint Louis, MO, USA). Rabbit (#A0545) and goat (#A4174) peroxidase-conjugated secondary antibodies, media and sera for cell cultures were purchased from Sigma-Aldrich; mouse peroxidase-conjugated secondary antibody (#sc-2005) was from Santa Cruz Biotechnology, Inc. Plasticware was obtained from Corning Inc. (Corning, NY, USA). 2-deoxy-[3H]-D-glucose was provided by Perkin Elmer (Waltham, MA, USA). Other reagents for cell culture and microscopy were obtained from Sigma-Aldrich, except where otherwise indicated.

Protein samples were extracted from cells in RIPA buffer (Solarbio, Beijing, China) and analyzed in cells 24 h after irradiation with or without FAK knockdown. Total protein samples were blotted with the following antibodies: anti-FAK, anti-phospho-FAK (Y397), anti-phospho-paxillin (Y118), and anti-β-actin (GeneTex, Irvine, CA).