Anti β actin sc 47778

Protein lysates were obtained by homogenizing paravertebral muscle with lysis buffer. The protein concentration was measured using the Bio-Rad protein assay (Bio-Rad, Richmond, CA, USA). Approximately 50 µg protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% dried milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h, the membranes were subsequently incubated overnight with primary antibodies diluted in 5% dried milk-TBS containing 0.1% Tween-20. The primary antibodies were as follows: anti-AMPK (#2532, 1:1,000), anti-phosphorylated (p-)AMPK (:#2531, 1:1,000) (both from Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (sc-8035, 1;10,000), anti-β-actin (sc-47778, 1:10,000) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-SIRT3 (ab86671, 1:500), anti-MnSOD (ab13534, 1:2,000) and anti-PGC1α (ab54481, 1:1,000) (all from Abcam Cambridge, MA, USA) antibodies. The membranes were then incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (sc-2005 and sc-2003, Santa Cruz Biotechnology, Inc.). An enhanced chemiluminescence system was used to visualize the bands. Densitometric analysis was performed using a gel image analysis system (UVP Inc., San Gabriel, CA, USA).

The whole-cell protein extracts were prepared by lysing the cells with 2% CHAPS lysis buffer in the presence of 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM EDTA and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The extracted proteins were resolved on a 4-12% polyacrylamide SDS-PAGE, as previously described (23 (link),24 (link)). The primary antibodies used were as follows: anti-RET (#3220), anti-RET/PTC1 (#14698), anti-cMYC (#5605), anti-p-mTOR (#2971) and anti-mTOR (#2972) (dilution 1:1,000) were purchased from Cell Signaling Technology (Beverly, MA, USA), anti-Bcl-2 (sc-7382), anti-pERK (sc-7383), anti-ERK (sc-271270), anti-cyclin D1 (sc-20044) and anti-β-actin (sc-47778) (dilution 1:300) were purchased from Santa Cruz Biotechnology. Anti-VEGF antibody was purchased from GeneTex (Irvine, CA, USA; #GTX102643). Mouse and rabbit IgG antibodies tagged with horseradish peroxidase (HRP) (Bio-Rad Laboratories, Hercules, CA, USA) were used as secondary antibodies (dilution 1:1,000). An enhanced chemiluminescence substrate kit (#32106) purchased from Thermo Fischer Scientific was used for detection.