Bca protein quantification kit

Cells were treated with Res for 48 h, and then washed with ice-cold phosphate-buffered saline (PBS) three times and then lysed by RIPA buffer containing protease and phosphatase inhibitors. The protein concentrations were detected by bicinchoninic acid (BCA) protein quantification kit (Beyotime, P0012, Shanghai, China). Western bloting was performed by the method described elsewhere 31 (link). Briefly, proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked by 5% skimmed milk in Tris-buffered saline (TBS-T) for 3 hours, followed by incubated with the primary antibody (rabbit anti-human NIS polyclonal antibody, 1:600; rabbit anti-human PTEN polyclonal antibody, 1:1000; rabbit anti-human E-cadherin polyclonal antibody, 1:1000, Proteintech, 20874-1-AP, Chicago, USA; rabbit anti-human GAPDH polyclonal antibody, 1:2000, Wanleibio, WL01547, Shenyang, China) overnight at 4°C. The next day, the primary antibody was discarded, and then the membrane was washed three times by TBST, followed by 1h incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG. The bands were visualized by the ECL system (Amersham Imager600, GE Healthcare Life Sciences, USA). The labeling signal was removed with a stripping buffer, and the membrane was incubated with another primary antibody until all the parameters were examined.

Protein extraction was proceeded in refrigeration room to avoid protein digestion. The tissues were first washed by phosphate-buffered saline (PBS) and quickly dissected using surgical scissors. Then the total protein was extracted by 8 M Urea in 100 mM NH₄HCO₃ (pH 8.0) containing Protease Inhibitor (Roche) and Phosphatase Inhibitor (Roche) on ice for 30 min, followed by 3 min sonication under the condition of 3 s on and 5 s off with 30% power of JY92-IIN (NingBoXinZhi, China). Finally, the protein solution was collected after centrifugation, and the concentration was measured by BCA protein quantification kit (Beyotime Biotechnology, China).

Whole cells were lysed in cell lysate buffer (PMSF:RIPA=1:100, Beyotime, China), and protein concentrations were quantified with a BCA protein quantification kit (Beyotime, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in fat-free milk and incubated with specific antibodies at 4 °C for 12 hours. The antibodies included anti-PHLPP2 (Abcam, USA), anti-CD133 (Proteintech, USA), anti-CD44 (CST, USA), anti-EPCAM (CST, USA), anti-Nrf2 (CST, USA), and anti-GAPDH (Hangzhou Xianzhi, China). Membranes were then incubated with secondary antibody (ZSGB-bio, China) and detected using a chemiluminescence system (Bio-Rad).

Cellular protein was lysed by radio immune precipitation assay (RIPA) buffer (Beyotime, China) with phenylmethane sulfonyl fluoride (PMSF) protease inhibitor (Beyotime, China) and the homogenate was centrifuged at 12,000 × g for 5 min at 4 ^◦C. The supernatant was collected and the protein concentration was determined immediately using a BCA protein quantification kit (Beyotime, China). The proteins were separated in 10% SDS-PAGE and transferred onto PVDF membrane, and then probed with antibodies following standard procedures. The antibodies and their dilutions utilized for western blots were as follow: anti-GHR (bs-0654R; Bioss, China; 1:500), anti-PGC1α (bs-1832R; Bioss, China; 1:500), anti-NRF1 (12,482–1-AP; Proteintech, USA; 1:500), anti-TOMM20 (AF1717; Beyotime, China; 1:500), anti-JAK2 (bs-0908R; Bioss, China; 1:1000), anti-p-JAK2 (bsm-52171R; Bioss, China; 1:1000), anti-AKT1 (bs-0115 M; Bioss, China; 1:500), anti-p-AKT1 (66,444–1-Ig; Proteintech, China; 1:500), anti-CREB1 (bs-0035R; Bioss, China; 1:500), anti-p-CREB1 (bs-0036R; Bioss, China; 1:500), anti-β-actin (bs-0061R; Bioss, China; 1:1000), goat anti-rabbit IgG-HRP (bs-0295G; Bioss, China; 1:5000), goat anti-mouse IgG-HRP (bs-0296G; Bioss, China; 1:5000).

The transfected cells were lysed in protein lysis buffer (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China) on ice for 30 min. The supernatants were collected after centrifugation for 15 min at 14,000 r/min and the concentration of the protein was calculated by the BCA Protein Quantification Kit (Beyotime Institute of Biotechnology). Proteins were separated by 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) in transfer buffer at 300 mA for 2 h. Then the membranes were incubated with Tris-buffered saline (TBS) containing 5% non-fat milk powder for 2 h at 4°C. The membranes were washed with TBS containing 0.2% Tween- 20 (TBST) three times, a nd then incubated with primary antibodies at 4°C overnight. Later, the membranes were washed with TBST three times and the specimens were hatched with secondary antibody in a secondary antibody solution for 2 h at room temperature. The indicated proteins were detected using a horseradish peroxidase chemiluminescent kit (Thermo Fisher Scientific) using an optional CCD camera and image processing system (Bio-Rad, Hercules, CA, USA). β-actin was used as a loading control. Moreover, N-cadherin, Vimentin, MMP9, AKT, phospho-AKT, mTOR, phospho-mTOR were bought from Cell Signaling Technology, Danvers, MA, USA. The secondary antibodies were also from Cell Signaling Technology.