Bca protein determination method

The supernatant of colonic tissues used for western blotting was prepared as described for ELISA. The concentration of protein was determined by the classical BCA protein determination method (Beyotime, Nanjing China). The same amount of protein was separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After sealing with 5% skimmed milk, the membranes were incubated with primary antibodies overnight at 4°C. The antibodies were anti-GAPDH (1:3,000), phosphoinositide 3-kinase (PI3K) (1:2,000), Akt (1:2,000), phospho (p)-Akt (1:1,500), Id2 (1:1,000), T-bet (1:2,000), FOXO3a (1:1,000), Noxa (1:2,000), C-myc (1:2,000), Rictor (1:2,000), Raptor (1:2,000), tuberous sclerosis complex (TSC)1 (1:1,000), TSC2 (1:2,000), p-AMP-activated kinase α (1:2000), AMPKα (1:2,000), eukaryotic translation initiation factor 4E-binding protein (4E-BP)2 (1:1,000), kinesin family member (Kif)2a (1:1,500), and 70-kDa ribosomal protein S6 kinase (p70S6K) (1:2,000). All primary antibodies were purchased from Abcam (Cambridge, UK). Then the membranes were washed with tris buffered saline tween (TBST) and incubated with the secondary antibody (1:2,000–1:4,000) (Abcam) at 37°C for 1 h. After washing with TBST again, the labeled protein bands were scanned with HP Scanjet 5500 (Hewlett Packard France, Les Ullis, France).

Tissue proteins were extracted by RIPA lysis buffer (Beyotime, China) containing protease inhibitors to preserve target proteins. The concentration was detected by the BCA protein determination method (Beyotime, China). The proteins were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred by using the polyvinylidene fluoride (PVDF) membrane, and blocked with 5% BSA for 60 min. The membrane was incubated with primary antibodies at 4°C overnight, including α-SMA (1 : 1000) (Abcam, UK), collagen III (1 : 1000) (Abcam, UK), RhoA (1 : 1000) (CST, US), ROCK1 (1 : 1000) (Abcam, UK), and p-MLC (1 : 1000) (CST, US). Then, the membrane was soaked with secondary antibodies at room temperature. The membrane was automatically visualized in the gel documentation analysis system, and those images were assembled by Image Lab software.