Anti fam134b

Cells were seeded onto coverslips 48 h before treatment and then permeabilized with blocking buffer [0.05% (w/v) saponin, 0.5% (w/ v) BSA, 50 mM NH 4 Cl, and 0.02% NaN 3 in PBS, pH 7.4] for 30 min, fixed in 4% paraformaldehyde for 30 min, and incubated with 5% BSA for 1 h. Then, the cells were incubated overnight at 4°C with the following primary antibodies: anti-FAM134B (1:1000 dilution; Abcam, Cambridge, UK), anti-cytochrome C (1:1000 dilution; Abcam), anti-IP3R (1:1000 dilution; Abcam), and washed three times with PBS. After incubation for 1 h with secondary antibodies (Alexa Fluor-labelled goat anti-rat A11077, goat anti-rabbit A11011/ A11008, and goat anti-mouse A11001; Thermo Scientific), the cells were washed with PBS three times and then incubated for 30 min with 1 μM ER tracker (Invitrogen, Carlsbad, USA). The cells were subsequently mounted onto Vectashield (Vector Laboratories, Newark, USA) and treated with 4′,6-diamidino-2-phenylindole (DAPI). The slides were observed under a fluorescence microscope (EVOS FL Auto Imaging System; Life Technologies).

Proteins were extracted from frozen placental tissues and HTR8/ SVneo cells with RIPA lysis buffer (Beyotime, Shanghai, China). Following the manufacturer's protocol, the protein concentration was determined using a BCA Protein Assay kit (Beyotime). Western blot analysis was performed based on the technique established in our laboratory [26] . As previously described, protein samples were subjected to SDS polyacrylamide gel electrophoresis, resolved by electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA) [27] (link). The antibodies used were anti-FAM134B (1:1000 dilution; Abcam), anti-IP3R (1:1000 dilution; Abcam), anti-FACL4 (1:1000 dilution; Abcam), anti-cytochrome C (1:500 dilution; Abcam), anti-cleaved caspase-3 (1:1000 dilution; Abcam), anti-IP3R (1:1000 dilution; Abcam), anti-calnexin (1:1000 dilution; Abcam), anti-BNIP3 (1:1000 dilution; Abcam), anti-PSME2 (1:1000 dilution; Abcam), and anti-β-actin (1:1000 dilution; Abcam). All incubation was performed at 4°C overnight. The polyvinylidene difluoride membranes were then incubated with a secondary antibody conjugated with horseradish peroxidase (1:5000 dilution; Abcam). The protein bands were visualized using western bright ECL kit (Advansta, Menlo Park, USA) and quantified with the ChemiDoc™ XRS+(Bio-Rad, Hercules, USA).