Rip3 antibody

Protein concentration in the hippocampus was determined as in our previous study. A quantity of 20–40 μg of protein was loaded onto an 8–15% gradient polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane and probed with the following primary antibodies: RIP1 antibody (1:1000, Abcam, United States); RIP3 antibody (1:1000, Abcam, United States); neuroligin (1:500, Abcam, United States); neurexin (1:500, Abcam, United States); PSD95 (1:1000, Cell Signaling); CREB (1:1000, Cell Signaling); phospho-CREB (p-CREB) (1:1000, Cell Signaling); BDNF (1:1000, Santa Cruz Biotechnology); β-actin (1:2000, Sigma-Aldrich) was used as an internal control. Secondary antibodies were horseradish peroxidase conjugated to mouse anti-rabbit/mouse immunoglobulin G (1:10,000, Sigma-Aldrich).

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.