Secondary antibodies antimouse and antirabbit

The MCF-7 and HCT-116 cells were lysed in ice-cold NP40 lysis buffer containing protease cocktail-inhibitor (Sigma-Aldrich). The protein concentrations were calculated using the Bradford method (Bio-Rad). Then, 30 μg protein were loaded and separated by 12% SDS-PAGE and then blotted onto a nitrocellulose membrane (Bio-Rad). The membrane was then blocked with 5% skimmed milk powder, then washed with Tris-buffered saline, 0.1% Tween 20 (TBST), and incubated at 4 °C overnight with primary IgG-unlabeled antibodies, anti-survivin (EPR2675) (ab134170); anti-CDK1 (A17) (ab18) and anti–cyclinB (Y106) (ab32053), purchased from Abcam, Cambridge, MA, USA. Secondary antibodies (antimouse and antirabbit; Cell Signaling Technology) were reacted for one hour with the membrane at 1:1000 dilutions. Chemiluminescence (Thermo Fisher Scientific) was used to detect the bands and Image Lab software (ChemiDoc Touch Gel and Western blot imaging system; Bio-Rad) was used to quantify the band-density; β-actin was used as a normalization control.

Equal micrograms of protein from the differential centrifugation pellet protein lysate (RIPA buffer) were mixed with non-reducing 4× LDS sample buffer (Thermo Fisher), heated at 98 °C for 10 min, and kept on ice briefly before loading on 10% Mini-protean TGX Precast gels (Bio-Rad) under non-reducing conditions. The proteins were transferred to Immobilon-P PVDF membranes (Millipore), which was then blocked with 5% non-fat milk in 1× TBST (1× TBS-0.1% Tween 20). Primary antibodies (1:1000) were incubated with membranes overnight at 4 °C, and secondary antibodies, anti-mouse, and anti-rabbit (1:2000, Cell Signaling) were incubated at room temperature for 1 h. Development was performed using SuperSignal^TM West Pico Plus Chemiluminescent Substrate (Thermo Fisher) and detected by ImageQuant LAS 4000 (GE Healthcare Life Science).

An ice-cold NP40 lysis buffer containing protease cocktail-inhibitor (Sigma-Aldrich) was used to lyse both the carnosic acid-treated and untreated AGS cells. Thirty micrograms of protein was separated using 12% SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Skimmed milk was used to block the membrane, followed by washing with tris-buffered saline with 0.1% tween solution (TBST). The membrane was incubated with primary IgG-unlabeled antibodies of AKT1 (#A17909), Phospho-AKT-S473 (#AP0637), mTOR (#A2445), and Phospho-mTOR-S2448 (#AP0115), purchased from ABclonal technology (Woburn, MA, USA), and survivin (EPR2675) (ab134170) purchased from Abcam, Cambridge, MA, USA, at 4 °C overnight. Later, the membrane was incubated with secondary antibodies (anti-mouse and anti-rabbit; Cell Signalling Technology) at 1:1000 dilutions for one hour. Chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) and Image Lab software (ChemiDoc Touch Gel and Western blot imaging system; Bio-Rad) were used to detect the bands and quantify band-density, respectively. β-actin was used as a normalization control.