Ab137055

The slides containing the tissue samples were deparaffinized and rehydrated and then subjected to epitope retrieval according to routine methods. After endogenous peroxidase activity was blocked, the slides were incubated with 10% goat serum in PBS. The slides were then incubated with a primary rabbit monoclonal antibody against ATP1B3 (1:50; Abcam, ab137055, Shanghai, China) at 4°C overnight. The slides were washed three times with PBS and then incubated with an anti-rabbit secondary antibody for 2 h at 37°C. The tissue sections were stained with 3,3’-diaminobenzidine (ZSGB, Beijing, China) and haematoxylin. Finally, the slides underwent dehydration, clearing and mounting with neutral gums. The negative control samples were treated identically, but the rabbit antibody against ATP1B3 was replaced by PBS.
A combined scoring system, in which the staining intensity (SI) was multiplied by the percentage of positive cells (PP), was used to evaluate immunoreactivity for ATP1B3 protein expression. Scores from 0-3 were given for the SI or the PP, as follows: score of 0 (negative or < 5%); score of 1 (weak or 6-25%); score of 2 (moderate or 26-50%); score of 3 (strong or 51-70%); score of 4 (only 71-100%). Then, a final decision for ATP1B3 expression was made according to a standard scoring system (negative expression: score<4; expression: score≥4).

Total protein was isolated from tissue samples or cultured cells using RIPA lysis buffer (Beyotime, Shanghai, China) with a protease inhibitor cocktail and was then quantified by BCA assay (Beyotime, Shanghai, China). Equal amounts (20 μg) of protein samples were loaded onto a 12% SDS-PAGE gel and then transferred to PVDF membranes. The membrane was blocked by 5% skim milk in PBS at room temperature for 1 h and incubated with primary rabbit antibodies against human ATP1B3 protein (1:1000; Abcam, ab137055, Shanghai, China), PI3K (1:1000; Abcam, ab189403, Shanghai, China), AKT (1:500; Abcam, ab8805, Shanghai, China), p-AKT (1:1000; Abcam, ab38449, Shanghai, China), Caspase-3(1:5000; Abcam, ab32351, Shanghai, China) and GAPDH (1:1000; Bioworld Technology, Beijing, China) overnight at 4°C. After the membranes were washed with TBST, they were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2000; Abcam, ab6721, Shanghai, China) for 2 h at room temperature. Signals were detected by enhanced chemiluminescence (ECL) reagent (Biogot, USA).