P β catenin sc 16743 r

Crystal violet was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against AKT (#4685), phospho-AKT (Ser473) (p-AKT; #4060), phospho-mTOR (Ser2448) (p-mTOR; #5536), mTOR (#2983), phospho-S6 kinase (Thr389)(p-S6K; #9205), S6K (#2708), phospho-4EBP1 (The37/46) (p-4EBP1; #2855), 4EBP1 (#9644), cyclin D1 (#2926), GSK-3β (#9332), and phospho-GSK-3β (Ser9)(p-GSK-3β; #9336) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-vimentin antibody (ab92547) and anti-c-Myc antibody were obtained from Abcam (Cambridge, UK). Antibodies against phospho-β-catenin (p-β-catenin; sc-16743R) and β-catenin (sc-7199) were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against PGC-1α (A12348) was purchased from ABclonal Biotech Co., Ltd. (Boston, MA, USA). Antibody against LARS1 (21146-1AP) was purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Anti-β-actin (A1978), anti-rabbit IgG (A0545), and anti-mouse IgG secondary antibodies (A9044) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Aliquots of 30 µg protein extracts were subjected to SDS-PAGE electrophoresis. Proteins on the gels were blotted onto a nitrocellulose membrane. Proteins of interest on the blots were, respectively, probed by GSK3β (#9315, Cell signaling, Danvers, MA, USA), Dkk1 (sc-25516, Santa Cruz Biotechnology, Dallas, MA, USA), GAPDH (GTX100118, GeneTex, Irvine, CA, USA), phospho-Smad3 (p-Smad3; ab51451, abcam, Cambridge, UK), Smad 3 (ab40854, abcam, Cambridge, UK), phospho-β-catenin (p-β-catenin ; sc-16743-R, Santa Cruz Biotechnology, Dallas, MA, USA), and β-catenin (sc-7963, Santa Cruz Biotechnology, Dallas, MA, USA). After washing, the blots were further incubated with secondary antibodies horseradish peroxidase-coupled anti-mouse immunoglobulin-G antibodies. The proteins designated were visualized by an enhanced chemiluminescence detection (GE Healthcare Biosciences AB, Little Chalfont, UK). Intensities of protein bands were quantified by a gel densitometry system. Six animals were used for detection.