Anti akt

Proteins were extracted using RIPA buffer, and protein concentration was measured using BCA Kit (EnoGene, Nanjing, China). Proteins were separated by 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane (Bio-Rad, USA). The membranes were blocked with 5 % skimmed milk at room temperature for 2 h, and washed in TBS-Tween 20. Subsequently, the membranes were incubated with anti-ROR2 (Biovision, Cat. 6702-100), anti-Bax (EnoGene, Cat. E11-0132C), anti-Bak (EnoGene, Cat. E11-0131C), anti-Bcl-2 (EnoGene, Cat. E10-30077), anti-Bcl-xl (EnoGene, Cat. E90209), anti-mTOR (EnoGene, Cat. E11-7156B), anti-survivin 1 (Biorbyt, Cat. orb394299), anti-PI3K (Biorbyt Cat. orb137259), anti-AKT (bioss, Cat. bs-0115R-1), anti-pAKT (bioss, Cat. bs-12458R-1), anti-PDK1 (EnoGene, Cat. E10-30154), anti-p21 (Abcam, Cat. ab215971-p21), anti-Cyclin D1 (Abcam, Cat. ab185241 - Cyclin D1), and control anti-GAPDH (EnoGene, Cat. E12-052) antibodies at 4 °C overnight. After washing in TBS-Tween 20, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP, EnoGene) at 37 °C for 1 h. Protein bands were detected using ECL Western Blotting System (Millipore, MA, U.S.A.) and visualized using image analyzer (DKSH, USA). The immunoblot signal was quantitated with ImageJ software and the values were normalized to the GAPDH band density.

Proteins were extracted using radio immunoprecipitation assay lysis buffer with 1% phenylmethanesulfonyl fluoride (Beyotime). After measuring protein concentrations with a BCA protein assay kit (Beyotime), 30 μg proteins from each group were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States). Following blockade with 5% skim milk or 1% bovine serum albumin, the membranes were incubated with anti-cyclinB1, anti-cyclinD1 (1: 400; Boster), anti-PTCH (1: 500; Novus Biologicals, Littleton, CO, United States), anti-Smo (1: 100; Santa Cruz, Dallas, TX, United States), anti-Gli1 (1: 200; Santa Cruz), anti-AKT, anti-p-AKT (1: 500; Bioss, Beijing, China), anti-Shh (1: 1000; Abcam, Cambridge, United Kingdom) or anti-β-actin (1: 1000; Santa Cruz) antibodies at 4°C overnight. The membranes were rinsed in tris buffered saline with Tween (TBST) and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 5000; Beyotime) at 37°C for 45 min. Thereafter, the membranes were rinsed in TBST and visualized with an enhanced chemiluminescence detection kit (Beyotime). Relative protein levels were calculated using β-actin as the internal reference.