Anti gapdh

The antibodies used in the present study included the following: anti-SQSTM1/p62 rabbit mAb (ab109012) from Abcam, anti-LC3A/B (12741), anti-ATG7 (8558), anti-PD-L1 (13684 or 41726), anti-KRAS (3339), anti-p44/42 MAPK (Erk1/2) (4695), anti-Beclin-1(3495), anti-ATG5 (12994), and anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370) from Cell Signaling Technology, and anti-GAPDH from Yeasen. Annexin V-APC and 7-AAD apoptosis detection kit were purchased from Biolegend. Trametinib and SCH772984 were purchased from Selleck Chemicals (Houston, TX, USA).

Cultured cells were lysed with lysis buffer. Equal amounts of protein were run on 10 % SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5 % milk in TBST, membranes were incubated with primary antibodies overnight. The following antibodies were used: anti-ANT3 (1:1000, Proteintech, 14B41-1-AP), anti- FBLN1 (1:1000, Bioss, bs-0809R), E6/E7 (Abcam; ab70, ab30731) and anti-GAPDH (1:6000, Yeasen). Membranes were then incubated with the Rabbit peroxidase-conjugated secondary antibody (1:10,000, Abclonal). The blots were detected by sensitive chemiluminescence liquid (Yeasen), Biorad software was used to captured the images.

EC cells were lysed in RIPA buffer (Epizyme, PC102, Ipsen Biopharmaceuticals, Cambridge, MA, USA) supplemented with a protease inhibitor cocktail. Samples were then quantified using a BCA kit (Epizyme, ZJ101), resolved by SDS‐PAGE, and blotted with primary antibodies and corresponding horseradish peroxidase‐conjugated secondary antibodies. The primary antibodies used included anti‐AMPK (1:1000 dilution), anti‐p‐AMPK (1:1000 dilution), anti‐BIM (1:1000 dilution), and anti‐GRP75 (1:1000 dilution) from Cell Signaling Technology (Beverly, MA, USA) and anti‐GAPDH (1:1500 dilution) from Yeason.
Co‐immunoprecipitation of the MAM fractions was conducted using Protein A/G PLUS‐Agarose (Santa Cruz Biotechnology, sc‐2003). Isolated MAM pellets were resuspended in 0.1% NP40 lysis buffer. For pre‐clearing, 1 µg of the control IgG was incubated with 20 µL volumes of the beads and incubated with MAM lysate at 4 °C for 30 min. The lysate was centrifuged at 4 °C for 5 min at 1000 × g, and the supernatant was incubated with the antibodies at 4 °C overnight. After adding 20 µL of the resuspended beads at 4 °C for 30 min, the collected beads were washed, and the precipitated protein was examined by Western blotting.

Whole cell lysates were prepared in RIPA buffer (Thermo Scientific) with phenylmethylsulfonyl fluoride and protease inhibitors and centrifuged. Supernatants were aliquoted, mixed with loading buffer, resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidenedifluoride membranes (Millipore, Billerica, MA). After blocked with 5% skim milk in TBST (Tris-buffered Saline with Tween 20) at room temperature for 1 h, the membranes were incubated with different primary antibodies, including anti-ERK, anti-p-ERK, anti-P65, anti-p-P65, anti-cyclin D1 (1:1000,Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (1:5000, Yeason, China) in 5% milk/TBST buffer at 4 °C overnight, and then probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h. After washing with TBST, the membrane was developed with enhanced chemiluminescent plus substrate (Merck Millipore, Billerica, MA, USA) and the signal was recorded by Fluorchem E System (Protein Simple, Santa Clara, CA, USA).

The placenta tissues and HTR8/SVneo cells were homogenized and lysed in ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, MA, United States). The lysates were boiled and resolved using SDS-PAGE. SDS-PAGE–separated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, United States). Target proteins were probed with indicators following their primary antibodies, which were as follows: anti-LC3B, anti-Beclin1, anti-P62 (Cell Signaling Technology, MA, United States), anti-BAX (Abcam, Cambridge, United Kingdom), anti-GAPDH, anti–β-actin, and anti–β-Tubulin (YEASEN, Shanghai, China). After incubation with a secondary antibody, the bands were captured using an Amersham Imager 600 (GE, MA, United States).