Rabbit anti prb ser807 811

For immunostaining, tissues were fixed in Zn-formalin for 24 hr and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 min each, and then blocked in 5% donkey serum for 1 hr at room temperature, incubated with either Rabbit anti-BNIP3 antibody at 1:100 (A5683, Abclonal), Rabbit anti-NF-kB-p65 1:400 (8242S, Cell Signaling Technology), Rabbit anti-Phospho-c-jun 1:200 (3270S, Cell Signaling Technology), Rat anti-ki67 1:100 (14-5698-82, ebiosciences), Rabbit anti-pRB-Ser807/811 1:400 (8516, Cell Signaling Technology), anti-p21 1:1000 (ZRB1141, Sigma), Rat anti-Phospho H3 1:1000 (H9908, Sigma), or Goat anti-GFP (ab6673, Abcam) at 1:250 overnight at 4°C, washed, incubated with secondary antibody AF594-anti-rabbit (A-21207, Invitrogen), Alexa594 anti-rat (A-21209, Invitrogen), and Alexa488 anti-goat (11055, Invitrogen) antibodies at 1:200 for 1 hr at room temperature, counterstained with DAPI, washed, and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera using ×400 magnification. MFI was calculated by measuring average intensity over a fixed threshold for all images. NF-kB staining was imaged via Zeiss LSM880 confocal microscope using ×63 oil-immersion objective, and intensity was measured on 0.8 µm z-sections.

Cells were harvested, washed with PBS, and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1% SDS, 1% NP-40, and 1% Triton X-100) supplemented with 1 mM PMSF (Sigma) and a protease inhibitor cocktail (Roche) at 4°C for 20 min. The lysates were then centrifuged for 15 min at 12,000 rpm at 4°C. The supernatants were collected, and an equal volume of 2X Laemmli’s buffer was added. The sample was boiled for 5 min at 95°C. Proteins were resolved by 10 or 12.5% SDS-PAGE and then transferred to nitrocellulose membranes (Pall Corporation). Membranes were blocked with 5% non-fat milk in TBST (0.1% Tween 20) for 1 h before incubation with primary and secondary antibodies sequentially. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following antibodies were used: rabbit anti-FOP (Abcam, ab156013, 1:2,000), mouse anti-GFP (Santa Cruz, sc-9996, 1:5,000), rabbit anti-AURKA (Cell signaling Technology, 14475, 1:2,000), rabbit anti-Cyclin A2 (Abcam, ab18159, 1:10,000), rabbit anti-pCDC2 (Tyr15) (Cell Signaling Technology, 9111, 1:2,000), rabbit anti-pRb (Ser807/811) (Cell Signaling Technology, 8516, 1:2,000), and mouse anti-β-actin (Sigma, A5441, 1:5,000).