Gsk 3β

Tissues dissected from the ileum of the STZ-induced rats were lysed in 300 μl buffer containing 1% protease inhibitor solution, and then centrifuged to collect the soluble proteins. The protein content was determined using the BCA Protein Assay kit by spectrophotometer (Bio-Rad, Mississauga, ON, Canada). Protein samples (60 μg) were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose membranes53 (link). After blocking in 5% nonfat milk for 2 h at room temperature, membranes were incubated with the indicated primary antibodies against GSK-3β (Cat No. WL01593, Wanleibio, Shenyang, P.R. China), p-GSK-3β (Ser-9) (Cat No. ap0039, ABclonal Biotech Co., Ltd, Wuhan, Hubei, P.R. China), β -catenin (Cat No. 610154, BD Biosciences, San Jose, CA, USA), and TCF7L2 (Cat No. Ab76151, Abcam, Cambridge, MA, USA) diluted at 1:500 in PBS buffer 4 °C overnight. Membranes were then washed in phosphate-buffered saline with Tween 20 and incubated for 1h with the fluorescence-conjugated anti-rabbit IgG secondary antibody (1:10000). Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (Area × OD) for each group and normalized to β-actin (Santa Cruz)54 (link). The normalized expression levels of target proteins are presented as fold changes normalized to the control values51 (link).

Protein was extracted from cells and tissues using ice‐cold RIPA buffer as previously described.²⁰ Equivalent amounts of protein determined by Bradford method and separated by 10% SDS‐PAGE. The protein bands were electrotransferred to nitrocellulose (NC) membrane (PALL), blocked with 5% skim milk (BD) and subsequently incubated with the primary antibodies CRKL (1:2000, Santa Cruz Biotechnology), PI3K (1:500, Sanying), p‐Akt (1:800, pThr308/Ser473, Cell Signaling), GLUT1 (Glucose transporters1, 1:500, Wanlei Biotechnology), HKII (Hexokinase II, 1:500, Wanlei Biotechnology), GSK3β (Glycogen syntheses kinase 3β, 1:500, Wanlei Biotechnology), ACTB (1:5000, TransGen Biotech) and GAPDH (1:5000, Sanying) at 4°C overnight. After incubation with the secondary antibody at RT for 3 hours, protein bands were observed by ECL and quantified using the Bio‐Rad ChemiDoc™ MP system (Bio‐Rad).