Anti actb antibody

Total cellular proteins were extracted from tissues or cells, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Pall, New York, USA). Membranes were blocked with 5% nonfat milk in 1% Tween-PBS (PBST) and then probed overnight with anti-RFC4 rabbit polyclonal antibody (1:1000, Epitomics, Burlingame, CA, USA) or anti-ACTB antibody (1:1000, Proteintech, Chicago, IL, USA). After three washing steps of 10 min in PBST, membranes were incubated with species-appropriate fluorescently-conjugated secondary antibodies (1:10000 in PBST, LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. The immunoreactive signals were detected using the two-color fluorescent western blotting Odyssey infrared imaging system (LI-COR Biosciences).

Whole cells were lysed in RIPA lysis buffer at 4 °C for 30 min. Proteins (20 µg of protein/lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes using a semi-dry transfer system (Bio-Rad). The membrane was blocked in 5% non-fat milk and incubated with anti-CCND1 C-terminus antibody (Cell Signaling), anti-CCND1 full length antibody (Thermo Fisher Scientific) or anti-Actb antibody (Proteintech) overnight at 4 °C. Then, the membrane was washed with TBS-T and incubated with corresponding horseradish-peroxidase-labelled secondary antibodies (Cell signaling) for 1 h. Protein signals were detected by supersignal west femto maximum sensitivity substrate (Thermo scientific).

The total proteins from the EECs were extracted using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The BCA Protein Assay Kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China) was used to measure total protein concentration. 30 μg of protein were fractionated on 12% SDS-PAGE gel, transferred to PVDF membranes and blocked in 10% nonfat milk in Tris-buffered saline containing 0.5% Tween-100 (TBST). The PVDF membranes were incubated with anti-BCL2L15 (BIOSS bs-7582, 1:500), anti- phospho-STAT3 (Ty705) (CST 9145, diluted 1:1000), anti-phospho-STAT1 (Ser727) antibody (CST 9177, diluted 1:1000), and anti-ACTB antibody (Proteintech Group, Inc, diluted 1:2000) overnight at 4 °C. After incubating with an HRP-labeled secondary antibody, the protein expression was visualized using the Image-Pro plus 6.0 software (Media Cybernetics, Inc., Silver Spring, MD, United States).