Mbs435036

Cells were collected after transfection. Overexpression rate above 200% compared with control cells was achieved through the detection of lncRNA GASL1 expression by qRT-PCR. Total RNA extractions from in vitro cultured cells were performed using RIPA buffer (Thermo Fisher Scientific Inc., Waltham MA, USA). After measurement of protein concentrations by BCA assay, protein samples were denatured at 95°C for 5 min, followed by 12% SDS-PAGE gel electrophoresis with 22 μg of protein in each well. Gel transfer was performed to VDF membranes (Thermo Fisher Scientific Inc., Waltham MA, USA). Membranes were incubated with 5% dried skimmed milk powder for 2h at room temperature. Then, the membranes were cultured with rabbit anti-human TGF-β1 primary antibody (1: 2000) (ab92486) (Abcam, Cambridge, MA, USA) and rabbit anti-human GAPDH primary antibody (1: 2000) (ab181602) (Abcam, Cambridge, MA, USA) overnight at 4°C. The membranes were further incubated with goat anti-rabbit IgG-horseradish peroxidase (HRP)-conjugated secondary antibody (1: 1,000) (MBS435036) (MyBioSource, San Diego, CA, USA) for 2 h at 25°C. Signals were developed by Pierce enhanced chemiluminescence (ECL) Western blotting substrate (Thermo Fisher Scientific Inc., Waltham MA, USA). Data normalization was performed using Image J 1.48 software National Institutes of Health (NIH) (Bethesda, MD, USA).

Total proteins in 3×10⁴ cells of each transfection group were isolated by RIPA solution (Sangon). BCA assay (Sangon) was performed to quantify protein samples. After proteins were denatured at 95°C for 10 min, proteins were separated by performing electrophoresis (12% SDS-PAGE gel). PVDF membrane was used to transfer proteins and 5% non-fat milk (PBS) was used for blocking. After that, membranes were first incubated with anti-GAPDH (1: 1000, ab37168, Abcam) and anti-TGF-β1 (1: 1000, ab92486, Abcam) rabbit primary antibodies for 18 h at 4°C. Following that, IgG-HRP (1: 1500, MBS435036, MyBioSource) goat secondary antibody was used to further incubation for 2 h at 24°C. Finally, ECL (Sigma-Aldrich, USA) was used for signal production and all data were normalized using Image J v1.48 software.

Conventional method was used to extract total protein from cultured cells, and protein quality was tested by BCA method. Then 10% SDS-PAGE gel electrophoresis was performed to separate protein, followed by transmembrane to PVDF. After blocking in 5% skim milk, membranes were washed and incubated with primary antibodies including rabbit anti-mTOR (1: 2,000, ab2732, Abcam), anti-phospho-mTOR (Ser2448) (1: 1,000, ab84400, Abcam), anti-GAPDH (1: 1,000, ab9845, Abcam) overnight at 4°C. After washing with PBS, anti-rabbit IgG-HRP secondary antibody (1: 1,000, MBS435036, MyBioSource) was added and incubated with the membranes at room temperature for one hour. After washing, signals were detected using ECL method (Sigma-Aldrich, USA). Relative expression levels of mTOR and p-mTOR were calculated using ImageJ according to endogenous control GAPDH.

Protein was extracted using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Inc.) and protein concentration was measured using a BCA assay. After denaturing at 95°C for 10 min, 20 µg protein was subjected to 12% SDS-PAGE gel electrophoresis. Following gel transfer to PVDF membranes, 5% skimmed milk was used to block the membranes at room temperature for 1 h. Membranes were then incubated with rabbit anti-human HIF1α (1:1,650; cat. no. ab2185; Abcam) and rabbit anti-human GAPDH (1:1,400; cat. no. ab8255; Abcam) primary antibodies overnight at 4°C. Membranes were incubated the next day with IgG-horseradish peroxide conjugated secondary antibodies (goat anti-rabbit; 1:1,500; MBS435036; MyBioSource, Inc.) at room temperature for 1 h. Signals were subsequently developed by ECL (Sigma-Aldrich; Merck KGaA). Data normalization was performed using ImageJ software (v.1.60; National Institutes of Health).

RIPA solution (Thermo Fisher Scientific) was used to extract total protein from cells cultured in vitro. BSA assay was performed to measure protein concentration. SDS-PAGE gel (10%) electrophoresis was then performed with 35 μg protein per lane, followed by gel transfer to PVDF membranes (Bio-Rad, USA). Blocking was then performed by incubating membranes with skimmed milk (5%) for 2 h at room temperature. Membranes were then incubated with primary antibodies, including rabbit anti-human HIF1α (ab2185, 1: 1200; Abcam) and GAPDH (ab9485, 1: 1400, Abcam) primary antibodies overnight at 4°C. On the next day, IgG-HRP secondary antibody (1: 1000, MBS435036, MyBioSource) was incubated with the membranes for 3 h at room temperature. Finally, signal development was performed using ECL (Sigma-Aldrich, USA), and HIF1α expression was normalized to GAPDH using Image J software.