Ab179463

A human mesangial cell line (HMC cell line, FH0241) was obtained from FuHeng Biology Co. (Shanghai, China). DHT (116064-77-8, purity ≥ 98%) was acquired from Krre Technology Co. (Beijing, China). Mannitol (69-65-8) was obtained from Macklin Biochemical Co. (Shanghai, China). The following antibodies: GAPDH (1:1000, ab8245), α-smooth muscle actin (1:1000, ab7817), Collagen I (1:1000, ab260043), Fibronectin (1:1000, ab2413), PI3KCA (1:1000, ab40776), PI3K (1:1000, ab191606), p-PI3K (1:1000, ab182651), AKT (1:1000, ab179463), and p-AKT (1:1000, ab192623) were bought from Abcam Co. (Cambridge, United Kingdom). Goat Anti-Rabbit IgG H&L (HRP, A0208), Rabbit Anti-Mouse IgG H&L (HRP, A0216), and Rabbit Anti-Mouse IgG H&L (Alexa Fluor 488, A0428) were purchased from Beyotime (Shanghai, China). DAPI Staining Solution (C1006) and Cell Counting Kit-8 (C0038) were also obtained from Beyotime (Shanghai, China). Glucose, Trypsin, Dulbecco’s Modified Eagle’s Medium (DMEM), and fetal bovine serum (FBS) were purchased from GIBCO. The Mouse Albumin ELISA Kit (ab207620) was purchased from Abcam.

To prepare the protein samples for loading, transfected H1975 and A549 cells or tumor tissues were lysed using a radio-immunoprecipitation assay. After quantification, 40 µg of protein samples were subjected to separation through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking in non-fat milk, the membranes were incubated in Tris Buffered Saline Tween (TBST) containing primary antibody against Ki-67 (ab92742; Abcam), matrix metalloproteinase 9 (MMP-9; ab38898; Abcam), HK2 (ab209847; Abcam), phosphorylated protein kinase B (p-AKT; ab192623; Abcam), AKT (ab179463; Abcam), p-mammalian target of rapamycin (p-mTOR; ab109268; Abcam), mTOR (ab32028; Abcam), or GAPDH (ab181602; Abcam) and then incubated with secondary antibody (ab205718; Abcam). The blots were visualized using a chemiluminescence kit (Merck Millipore, Darmstadt, Germany). GAPDH served as a control.

Following the treatment described previously, proteins in the HepG2 cells were isolated through treatment with RIPA buffer (Meilunbio, China) with phosphatase inhibitor cocktail I (Meilunbio, China) on ice. The proteins were separated by SDS–PAGE and transferred to nitrocellulose membranes (Millipore, Billerica, United States). After blocking, the membranes were incubated overnight with antibodies at 4°C and then with horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies. Antibodies against PI3K (ab191606), phosphorylated (p)-PI3K (ab182651), Akt (ab179463) and p-Akt (ab192623) antibodies were purchased from Abcam; antibodies against mTOR (66888-1-Ig), p-mTOR (67778-1-Ig), Bcl-2 (12789-1-AP), Caspase-9 (10380-1-AP), Bax (50599-2-Ig), and β-actin (66009-1-Ig), and the secondary antibodies for mouse (SA00001-1) and rabbit (SA00001-2) were purchased from Proteintech. Finally, the blots were visualized with a ChampChemi detection system (Sage Creation, China).

The cultured cells were taken for immunoblot analysis. After lysis and centrifugation, the sample supernatant was quantified by BCA protein analysis kit. Then, protein samples were separated by 10% polyacrylamide gel electrophoresis. Protein was transferred to polyvinylidene fluoride membrane and blocked with 5% skim milk to prevent nonspecific binding and then incubated overnight with appropriate antibodies and primary antibodies of internal reference at 4°C. After culture, the cell membrane was washed with TBST and then cultured with appropriate secondary antibody labeled with peroxidase. Compared with β-actin expression, all protein blots expressed average area density. Antibodies were sourced from Abcam, specific product information is anti-EGFR (ab52894), anti-EGFR (phosphoY1068, ab40815), anti-JAK1 (ab133666), anti-JAK1 (phosphoY1022 + Y1023, ab13805), anti-STAT3 (ab68153), anti-STAT3 (phosphoY705, ab76315), anti-PI3K (ab191606), anti-PI3K (phosphoY607, ab182651), anti-AKT1 (ab179463), and anti-AKT1 (phosphoS473, ab81283).

The PC cell lines were dissociated on ice for 30 min after supplementation of lysis buffer. After centrifugation at the speed of 12,000 rpm at 4°C, cell supernatants were collected for quantitation of protein levels. Total proteins (30 µg for each sample) were separated after undergoing sodium dodecyl sulfate (SDS-PAGE; BIO-RAD, USA), and the resultant samples were then transferred onto the polyvinylidene fluoride membrane. With 5% skimmed milk applied to block protein samples for 2 h, primary antibodies (Abcam, USA) against β-actin (rabbit anti-human, 1:1,000, ab8226), PI3K (rabbit anti-human, 1:1,000, ab191606), p-PI3K (rabbit anti-human, 1:1,000, ab182651), Akt (rabbit anti-human, 1:10,000, ab179463), p-Akt (rabbit anti-human, 1:500, ab38449), mTOR (rabbit anti-human, 1:10,000, ab134903) and p-mTOR (rabbit anti-human, 1:1,000, ab109268) were diluted to incubate the proteins at 4°C for overnight. After that the samples were rinsed with TBST which was inclusive of Tween 20, and then secondary antibodies labeled by horseradish peroxidase (1:5,000, Abcam, USA) were utilized to incubate protein samples for 2 h. Then the samples were monitored with electro-chemiluminescence in the darkness, and gray values of protein bands were analyzed utilizing ImageJ software.