Chemiluminescent peroxidase substrate 1

MDA-MB-231 cells were seeded (10⁵ cells/well) in a six-well plate in culture medium (DMEM) plus 10% FBS overnight at 37 °C and 5% CO₂ and then incubated with 10, 100 or 1000 nM ALT-C. After 24 h, culture medium was removed and cells were lysed with RIPA buffer [150 mM NaCl; 50 mM Tris; pH 8.0; 0.1% sodium dodecyl sulfate (SDS); 1% Triton Χ-100] and proteases and phosphatases inhibitors. Protein quantitation was carried out using the BCA Protein Assay kit (Thermo Scientific, USA), according to the supplier’s instructions.
Thirty micrograms of each sample were diluted in denaturing sample buffer containing glycerol, SDS, dithiothreitol (DTT) and bromophenol blue. After electrophoresis, the samples were transferred to nitrocellulose membranes and blocked with skimmed milk powder (4%). Rabbit monoclonal antibodies against p-FAK (ab81298) and FAK (ab40794) were used at 1:1000 dilution in PBS. A secondary anti-rabbit antibody (ab97051) was used at 1:10,000 dilution in milk powder. Detection of proteins was performed using the Chemiluminescent Peroxidase Substrate-1 (SLBJ1875, Sigma-Aldrich, USA). Images were obtained on a digital documentation system (Chemi-Doc Xr, Bio-Rad Lab, USA) and relative quantitation was done by densitometric analysis of the images using the Image J software and normalizing to GAPDH band densities when indicated.

Whole cellular extracts were obtained using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, and 0.1% SDS containing proteases and phosphatases inhibitors). Protein concentration was measured by the BCA protein assay and equal amounts were resolved by SDS-PAGE and transferred to Immobilon polyvinylidene difluoride (PVDF) membranes. Western blots analysis was performed using antibodies against phospho-Thr¹⁷²-AMPK, total AMPK, phospho-Ser⁴⁷³-AKT, total AKT (Cell Signaling), phospho-Ser⁷⁹-ACC, and total ACC (Upstate). Detection was performed using the appropriate horseradish peroxidase-labelled IgG and the Chemiluminescent Peroxidase Substrate-1 (Sigma). The size of detected proteins was estimated using protein molecular-mass standards (Thermo Scientific, Waltham, MA, USA). Western blot images were analyzed with a Molecular Imager (ChemiDoc XRS, BioRad) and quantified with Quantity One (BioRad).

Protein samples corresponding to 15 whole flies were prepared with RIPA buffer, supplemented with protease inhibitor cocktail (Roche). 50μg of total lysate as determined by Bradford assay (Sigma) was loaded and separated on a 10% acrylamide precast Novex gel (Invitrogen) under reducing conditions and transferred onto nitrocellulose membrane. Primary antibodies were incubated at 4°C overnight. Subsequently, species-specific HRP-conjugated secondary antibodies were used. Primary antibodies used are as follows: rabbit anti-P-Smad2 (Cell Signaling Technology, #3108) 1:1000; mouse anti-β-tubulin antibody 1:10 000 (Cell Signaling Technology, #4054). Secondary antibodies used were: goat anti-rabbit -HRP 1:5000 (Dako); anti-mouse-HRP 1:5000 (GE Healthcare). Blots were visualized by chemiluminescence using ECL (GE Healthcare) or Chemiluminescent Peroxidase Substrate-1 (Sigma).