Vinculin

Forty-eight hours after laser irradiation, nuclear proteins were extracted with RIPA buffer containing protease and phosphatase inhibitors (Roche). Protein concentrations of all samples were determined using Bradford method with Albumin Bovine Serum (Sigma-Aldrich). Ten Microgram of protein was loaded on Bis-Tris or Tris-Acetate gels (NuPAGE, Thermo Fisher Scientific) and transferred onto a PVDF membrane. After blocking with 5% non-fat dry milk, the membranes were incubated overnight on 4°C with primary antibodies against AKT (Cell Signaling Technologies), pAKT Ser473 (Cell Signaling Technologies), ERK 1/2 (Cell Signaling Technologies), pERK 1/2 Thr202/Tyr204 (Cell Signaling Technologies), and vinculin (Sigma-Aldrich); followed by incubation with secondary antibodies for 1 h. Protein bands were detected with enhanced chemiluminiscence (ECL), visualized with Image Reader LAS3000 and densitometry was performed with ImageJ. Protein levels were corrected to vinculin and their unphosporylated forms.

The primary antibody to survivin (AF886; R&D Systems, Wiesbaden, Germany) was used at a dilution of 1:400, to grp-78 (ab21685; Abcam, Cambridge, UK) at a dilution of 1:200, to calnexin (ab75801; Abcam) at a dilution of 1:1500 and to p-SMAD2/3 (SC-11769; Santa Cruz) at a dilution of 1:5000. As a loading control tubulin, vinculin or amido black staining were used. The antibodies to tubulin or vinculin (both Sigma-Aldrich, Munich, Germany) were used at dilutions of 1:10000 (tubulin) or 1:5000 (vinculin).

Protein concentrations were determined according to the Bradford method using BSA as standard. Cell lysates (10–20 μg per sample) were then separated by 5–20% SDS-PAGE with known molecular weight markers (Bio-Rad, California, USA) and transferred onto polyvinylidene fluoride membranes by standard procedures. Membranes were incubated in blocking solution (5% skim milk or BSA in TBS-T) for 3 h at room temperature, and hybridized overnight at 4°C with primary antibodies against vinculin (Sigma) (1:4000), AdipoR1 (Immuno-Biological Laboratories Inc., Minneapolis MN) (2 μg /ml), and also Akt (1:1000), phospho-Akt (s473 or t308) (1:1000), AMPK (1:1000), phospho-AMPK (1:1000) all from Cell Signaling Technology (CST, Danvers, MA, USA). The membranes were washed three times with TBS-T and then incubated with either anti-rabbit (Sigma) (1:10000) or anti-mouse (Abcam, Cambridge, U.K.) (1:10000) secondary antibodies for 1 h at room temperature. After washing, immunodetection was performed using an enhanced chemiluminescence solution (Clarity Max Western ECL Substrates, Bio-Rad) and scanned on Fujifilm LAS-3000 system and quantified using Image-J software (National Institutes of Health).