Anti phospho gsk3b

The cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche) and PMSF (Roche). Fifty micrograms of the protein extractions were separated by 10% SDS-PAGE transferred to 0.22 mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies.The autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). Anti-EZH2 was from Abcam (Hong Kong, China). Anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK, anti-phospho-GSK3b and anti-GSK3b were from Cell Signaling Technology (Boston, MA, USA). Anti-HOXB7 was from Abcam. Anti-p53 was from Santa Cruz Biotechnology (Dallas, TX, USA). Results were normalized to the expression of GAPDH (Rabbit anti-GAPDH).

Most chemicals used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA) including the followings: glucose, palmitate, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoilium bromide (MTT), DL-fluorocitric acid barium salt, Oil Red O and Eosin Y. Nile Red and Hematoxylin. BODIPY™-palmitate (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-caspase 3, anti-phospho-AKT, anti-total AKT, anti-phospho-GSK3b, anti-total GSK3b, anti-phospho-JNK, anti-total JNK, anti-phospho-P65, and anti-total P65 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-actin and anti-tubulin antibodies were purchased from Bethyl Laboratory (Montgomery, TX, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. The catalog numbers of all reagents and antibodies were listed in Supplementary Table 2 of SI.

Differentially treated cells were harvested, and 3 to 10 µg of protein per sample was used for Western blotting as described before [63 (link),97 (link)]. Used primary antibodies were anti-phospho-GSK3b (1:250, rabbit; #9336, Cell Signaling, Danvers, MA, USA), anti-phospho-Akt (1:250, rabbit; #4060, Cell Signaling), anti-phospho-mTOR (1:250, rabbit; #2971, Cell Signaling), anti-phospho-4E-BP1 (1:1000, rabbit; #2855, Cell Signaling), and anti-phospho-P70S6K (1:1000, rabbit; #9205, Cell Signaling) in 5% bovine serum albumin/tris-buffered saline with 0.1% Tween 20 (TBST). Secondary antibody was donkey-anti-rabbit IgG-HRP (1:12,500; A16035, Thermo Fisher Scientific) in 2% (w/v) casein/TBST. Loading of equal amounts of protein was confirmed by stripping and incubation of the membranes with anti-GAPDH (1:200, mouse; sc-47724, Santa Cruz Biotechnology, Dallas, TX, USA) in 2% (w/v) casein/TBST and the secondary antibody donkey-anti-mouse IgG-HRP (1:10,000; A16011, Thermo Fisher Scientific) in 2% (w/v) casein/TBST. The signal densities were quantified using ImageJ^® software. The signals were normalized to GAPDH, and the n-fold signal induction was determined in relation to the respective control samples (control = 1).