Antibodies specific for β actin

Fibroblasts were lysed and collected in cold lysis buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS 50 mM Tris pH 8.0) containing Halt protease inhibitor (ThermoFisher). Samples were sonicated for 5 minutes in a chilled water bath and centrifuged at 14,000 × g at 4 °C for 15 minutes. Supernatant was collected and protein concentrations were quantified using the Pierce BCA Assay (ThermoFisher) according to the manufacturer’s protocol. Proteins were separated on a 12% Bis-Tris Plus gel (ThermoFisher) and transferred to a PDVF membrane using the iBlot dry blotting system (ThermoFisher). Membranes were blocked for 1 hour with Odyssey buffer (LI-COR) and reacted overnight at 4 °C with antibodies specific for β-actin (Sigma, St. Louis, MO) and GAPDH (Cell Signaling, Danvers, MA) both diluted 1:1000 in blocking buffer. After washing in PBS to remove the primary Abs, membranes were incubated with secondary antibodies conjugated to infrared dyes 700 and 800 (Rockland) for 1 hour at room temperature. Immunoreactive blots were visualized and densitometry was performed using the Odyssey Infrared Imaging system (LI-COR).

After 72 hours of exposure to specified treatment, cells were lysed in 9 M Urea, 0.075 M Tris, pH 7.6. Protein concentration was determined by using the Bradford assay, and lysates were subjected to reducing SDS/PAGE by standard methods. Western blots were incubated with primary antibodies against AXL (R&D Systems; 1:1000) and phospho-AXL (Cell Signaling; 1:500). To confirm equal protein loading, blots were probed with antibodies specific for β-actin (Sigma Aldrich; 1:3000). Horse-radish peroxidase-conjugated secondary antibodies were used to detect protein expression (Jackson ImmunoResearch, West Grove, PA) and chemiluminescence was measured on a ChemiDoc (Bio-Rad Laboratories).