Bca protein assay reagent kit

Cell transfections were performed using Megtran1.0 (Origene, Maryland, USA) according to the manufacturer’s instruction. In brief, cells grown in six-well plates were transfected with mir-4271 mimics, mir-4271 inhibitors (Supplemental Table 5) or random mir-4271 to a final concentration of 100 nM. 24 h after transfection, cells were harvested and homogenized with lysis solution (50 mM Tris-Cl, pH 8.0; 150 mM NaCl; 0.02% sodium azide; 0.1% SDS; 1 μg/ml aprotinin; 1% Nonidet P-40; and 0.5% sodium deoxycholate) containing protease inhibitors (100 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin). Supernatant was collected after centrifuging at 12,000 g for 20 min at 4 °C. The BCA protein assay reagent kit (Boster, China) was used for the protein concentration determination. Lysates were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% nonfat milk, blots were probed with APOC3 antibody (Santa Cruz Biotechnology, USA, sc-50377, lot#3423) and incubated with a peroxidase-conjugated secondary antibody. Bands were visualized by enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL) and quantified by densitometry.

Cells grown in 6‐well plates were transfected with SOX17 constructs or empty vector pcDNA3.1 using Lipofectamine™ 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Cells were harvested 48 hours after transfection and lysed with lysis solution (50 mmol/L Tris‐Cl, pH 8.0; 150 mmol/L NaCl; 0.02% sodium azide; 0.1% SDS; 1 μg/mL aprotinin; 1% Nonidet P‐40; and 0.5% sodium deoxycholate) containing protease inhibitors (100 μg/mL phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, 2 μg/mL leupeptin). After centrifuging at 12,000 g for 20 minutes at 4°C, supernatant was collected and protein concentrations were measured using the BCA protein assay reagent kit (Boster, China). Lysates were resolved by 10% SDS‐PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% non‐fat milk, blots were probed with RIP3 antibody (Abcam, ab56164) and incubated with a peroxidase‐conjugated secondary antibody. Bands were visualized by enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL) and quantified by densitometry.