Cellytic mt mammalian tissue lysis reagent

Prostate cancer cells were prepared in lysis buffer (CelLytic MT Mammalian Tissue Lysis Reagent, C3228, Sigma-Aldrich), and protein concentration was quantified using a DC Protein Assay Kit II (5000112, Bio-RAD, Hercules, CA). Cell extracts (30 μg of protein per lane) were loaded and separated on SDS-PAGE (4–20% Bis-Glycine gradient gels, EC6025BOX, Invitrogen) and transferred to a PVDF membrane. Membranes were incubated with 5% milk for 1 h and incubated with anti-Tyro3 (585S) -Axl (4977), -Mer (4319), -GAPDH (2118), -P-4E-BP1 (2855), -4E-PB1 (9644), -P-p70S6K (9234), -p70S6K (2708), -P-AKT(S473) (9271), or -AKT(S473) (9272) antibody (all primary antibodies were obtained from Cell Signaling Technology, Danvers, MA) overnight at 4°C. Primary antibody was used with 5% dry milk. Blots were incubated with peroxidase-coupled secondary antibodies (W4011, Promega, Madison, WI) for 1 h at a ratio of 1:3000. Protein expression was detected with SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Scientific, Rockford, IL). The densitometric analysis of the Western blot were performed with ImageJ software (version 1.50i; National Institutes of Health, Bethesda, MD).

Human FST was quantified from the muscles of SCID/mdx mice by an enzyme-linked immunosorbent assay (ELISA) with a human FST-specific immunoassay kit (Quantikine; R&D Systems, Minneapolis, USA), according to the manufacturer’s protocol. Briefly, total soluble protein was extracted from 50 mg gastrocnemius, quadriceps, and/or tibialis anterior muscle with CelLytic MT Mammalian Tissue Lysis reagent (Sigma, St. Louis, Missouri, USA). A total of 100 μl of lysate was loaded per well, and muscle FST concentrations were determined against a standard curve made with recombinant human FST provided by the manufacturer.

Protein samples were obtained by homogenizing brain cortices of mice in CelLytic^™ MT mammalian tissue lysis reagent (Sigma, C3228) mixed with protease inhibitor cocktail (Sigma, P3840) and phosphatase inhibitor cocktail 2 (Sigma, P5726). The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C. The concentration of the protein was quantified by BCA assay. After being electrophoresed on 12% SDS-PAGE gel, the proteins were transferred onto FluoroTrans^® W PVDF membranes (Pall, 20685) via electrophoretic transfer system (Bio-Rad). Then the membranes were blocked with 5% skim milk in PBST for 1 hr followed by incubation with respective primary antibodies at 4°C overnight. After thoroughly washed with PBST, the membranes were further incubated with respective horseradish peroxidase conjugated secondary antibodies. Thereafter, the protein bands were visualized with ECL-prime kit.