Enhanced pierce ecl western blotting substrate

Cell treatment, protein extraction and quantification were performed as aforementioned. Normalized amounts of protein (approximately 30 µg each lane) were added to 12% SDS-PAGE gels, then electrically separated and transferred onto PVDF membranes (EMD Millipore). Membranes were blocked with 5% skimmed milk for 1 h at 37°C and stained with rabbit anti-phospho(p)-p70S6K (cat. no. 9205S; dilution 1:400), mouse anti-AKT (cat. no. 2920S; dilution 1:500), mouse anti-p-AKT (cat. no. 4051S; dilution 1:400) (all from Cell Signaling Technology, Inc.), mouse anti-p70S6K (cat. no. 611260; dilution 1:800) and mouse anti-Bcl-2 (cat. no. 51-6511GR; dilution 1:500) (all from BD Biosciences). Mouse anti-β-actin antibody (cat. no. HC201; dilution 1:1000; Beijing Transgen Biotech Co., Ltd.) was used as the loading control. Horseradish peroxidase-conjugated anti-rabbit antibody (cat. no. HS101-01) and anti-mouse antibody (cat. no. HS201-01) (both dilution 1:5,000; Beijing Transgen Biotech Co., Ltd.) were used as secondary antibodies. Specific proteins were detected using an enhanced Pierce ECL western blotting substrate (Thermo Fisher Scientific, Inc.). Grayscale values were measured using ImageJ v1.51s software (National Institutes of Health).

NCI-H1975 and PC9 cells (5×10⁵) were transfected with 10 nM siROR1 or siControl and then serum-starved for 12 h. At 72 h after siRNA transfection, cells were lysed with 300 μl of lysis buffer (Beyotime, Shanghai, China). All protein extraction buffers were supplemented with protease inhibitor cocktail (Millipore, Bedford, MA, USA) and phosphatase inhibitor (Roche, Basel, Switzerland). The protein concentration of each cell lysate was determined using the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Normalized amounts of the lysate samples were loaded and electrophoresed on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Immunoblotting was performed using antibodies detecting mTOR, phospho-mTOR, PTEN, phospho-PTEN, AKT, phosphor-AKT (Cell Signaling Technology, Danvers, MA, USA) with β-actin (TransGen, Beijing, China) used as loading control. HRP conjugated anti-mouse IgG (TransGen, Beijing, China) or anti-rabbit IgG (Abcam, Cambridge, UK) was used as secondary antibodies. An enhanced Pierce ECL Western Blotting Substrate (Thermo Scientific, Pittsburgh, PA, USA) was used to detect chemiluminescence.

U87 cells were harvested and split using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Tulsa, OK, USA) according to the manufacturer’s instructions. The protein concentration was determined using the Bio-Rad protein assay kit (Bio-rad, California, USA). The proteins in the cell lysates were separated using 7–12% SDS-PAGE gels, and then, they were electrotransferred onto a PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA). The protein bands were detected using the enhanced Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Oklahoma, USA) and quantified as the ratio to the α-tubulin levels. The quantification was performed using an ImageQuant 350 analyzer (Amersham Biosciences).