Primary antibodies against β actin

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phalloidin tetramethylrhodamine B isothiocyanate, bovine serum albumin (BSA) and dimethylsulfoxide (DMSO) were obtained from Sigma (St. Louis, MO, USA). Primary antibodies specific to Cdc42, Rac1, and RhoA were obtained from Abcam (Cambridge, UK), while the primary antibodies against β-actin, the secondary antibody goat anti-rabbit IgG/HRP, and propidium iodide (PI) were purchased from Cell Signaling Technology (Danvers, MA, USA). All other reagents used in the experiments were high-grade, commercially available products. ASX was isolated from L. vannamei as described previously [15 (link)22 ]. The extraction yield was 20.39 ± 1.02 mg of ASX per gram of shrimp shell, and identification of ASX was previously reported [15 (link)22 23 ]. ASX was dissolved in DMSO and applied to cultures at final concentrations of 0.25-1 µg/mL. The final concentration of DMSO in the culture medium was always less than 0.1% (v/v), which was non-toxic to the cell cultures.

Cells were lysed in RIPA buffer (Beyotime, Haimen, China) containing protease and phosphatase inhibitors (Roche). Equal amounts of the released protein were separated by SDS-polyacrylamide gel after denaturation, electroblotted onto a polyvinylidene difluoride membrane, and incubated for 1 h in 5% bovine serum albumin at room temperature. The blotted membranes were then exposed to primary antibodies overnight at 4°C and visualized with HRP-conjugated secondary antibody for 1 h at room temperature. The blotted membranes were developed with chemiluminescent reagents (Millipore, Billerica, MA, USA) according to the instructions provided by the manufacturer. Primary antibodies against β-actin, mTOR, phospho-Akt₄₇₃, Akt, phospho-S6, S6, Myc, and Hif-1α were obtained from Cell Signaling Technology. The blots were quantitatively analyzed using ImageJ software (X64, v. 2.1.4), and Akt, S6, or β-actin was used a control.

Primary antibodies against glucose transporter 1 (GLUT1; 1:2,000; cat. no. 12939), lysine-specific demethylase 1 (LSD1; 1:2,000; cat. no. 2184), HIF-1α (1:1,1000; cat. no. 36169), PHD2 (1:3,000; cat. no. 4835), AKT (1:2,000; cat. no. 2938) and phosphorylated (p)-T308-AKT (1:1,000; cat. no. 13038) were purchased from Cell Signaling Technology, Inc. Primary antibodies against β-actin (1:5,000; cat. no. ab16039), VEGF (1:1,000; cat. no. ab46154) and hydroxyproline (1:500; cat. no. ab37067; for detecting prolyl hydroxylated AKT) were purchased from Abcam. The horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. ab205718) was obtained from Abcam and normal rabbit IgG (1:500; cat. no. 2729) was purchased from Cell Signaling Technology, Inc. Vitamin C (100 µM; cat. no. 95209) and dimethyloxaloylglycine (DMOG; 1 mM; cat. no. D3695) were obtained from Sigma-Aldrich; Merck KGaA. The PHD2 selective inhibitor IOX2 (10 µM; cat. no. 9451) and AKT inhibitor A-674563 (5 µM; cat. no. B1761) were purchased from BioVision, Inc. The HIF-1 inhibitor BAY 87–2243 (100 nM; cat. no. B1115) was from APeXBIO Technology LLC and the PHD inhibitor Molidustat (5 µM; cat. no. S8138) was obtained from Selleck Chemicals. The above drugs were used to treat HLE-B3 cells at 37°C.

CFPAC-1 and AsPC-1 cells were harvested, washed, and lysed with RIPA buffer (Beyotime) containing protease inhibitor cocktail (Roche) for 30 min on ice. The total protein was extracted and quantified using a BCA kit (Beyotime) according to the manufacturer’s instructions. The protein samples were separated by electrophoresis using a 4%~20% gel and then transferred onto PVDF membranes (Millipore). The blotted membranes were blocked and incubated with primary antibodies overnight at 4°C. After 3 washes with TBST and a 1-h incubation with an HRP-conjugated secondary antibody (Proteintech) at room temperature, the membranes were developed using an enhanced chemiluminescence detection system (Bio-Rad Laboratories) to detect the protein. The primary antibodies against PC4 were obtained from Sigma, while the primary antibodies against β-actin, p-mTOR, t-mTOR, p-p70s6k, t-p70s6k, p-4EBP1, t-4EBP1, CDK4, CDK6, Cyclin D and Cyclin E were obtained from Cell Signaling Technology.