Rabbit anti pericentrin

The following primary antibodies were used: rabbit anti-centrin (Merck Millipore, MA, USA, 20H5) at 1:100, mouse anti-acetylated tubulin (Sigma, MO, USA, T7451) at 1:1000, rabbit anti-Cep63 (Merck Millipore, MA, USA, 06-1292) at 1:100, rabbit anti-Pericentrin (Abcam, Cambridge, UK, ab448) at 1:500, mouse anti-protamine (Novus Biologicals, CO, USA, H00005619) at 1:100, rabbit anti-β-tubulin (Abcam, Cambridge, UK, ab6046) at 1:1000, mouse anti-α-tubulin (Sigma, MO, USA, DM1A T6199) at 1:1000 in sperm and at 1:100 in oocytes and parthenotes, mouse anti-γ-tubulin (Sigma, MO, USA, GTU-448) at 1:100, rabbit anti-Poc1B (Thermo Fisher Scientific, MA, USA, PA5-24495) at 1:100, rabbit anti-WDR62 (Bethyl Laboratories, TX, USA, A301-560A) at 1:100, rabbit anti-Nek9 (Dr. Joan Roig gift) at 1:100, rabbit anti-Pontin (Home Made) at 1:100 and rabbit anti-Reptin (Home Made) at 1:100. Secondary antibodies anti-rabbit and anti-mouse conjugated to Alexa-488 and 568 (Invitrogen, CA, USA) were used at 1:1000 in sperm and cell culture, and 1:100 in oocyte and parthenotes for IF and 680 (Invitrogen, CA, USA) or IRdye 800 CW (LI-COR Biosciences, NE, USA) at 1:10 000 for western blots. Hoechst 33342 (1 µg/ml, Invitrogen, CA, USA) was used to visualize DNA.

Cells plated on coverslips were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton for 10 min at room temperature. The cells were then blocked with 3% bovine serum albumin (BSA) dissolved in PBS for 30 min and incubated with primary antibodies diluted at 1:200 with 3% BSA overnight. The cells were washed with PBS to remove the primary antibodies, followed by incubation with secondary antibodies diluted at 1:200 with 3% BSA for 1 hour. Immuno‐stained samples were then incubated with 0.1 μg/mL 4', 6‐diamidino‐2‐phenylindole (DAPI, Beyotime, Shanghai, China) diluted with PBS for 10 min. After washing with PBS, all immuno‐stained samples were observed and captured using the LSM880 system (Zeiss, Oberkochen, Baden‐Württemberg, Germany). The ZEN software (Zeiss) was used to process and analyze the images. Primary antibodies used were mouse anti‐α‐tubulin (Beyotime, Cat#AT819), rabbit anti‐pericentrin (Cat#ab4448, Abcam, Cambridge, MA, USA), mouse anti‐CENPA (Cat#GTX13939, Genetex, Irvine, CA, USA), rabbit anti‐AURKB (Cat#GTX132702, Genetex, Irvine, CA, USA), rabbit anti‐MCAK (Cat#12139‐1‐AP, Proteintech, Rosemont, IL, USA). Secondary antibodies used were Alexa Fluor 488, 546 (Invitrogen, Waltham, MA, USA).

Cells were cultured on chamber slides. For knockdown studies, SW480 and CACO2 cells were transfected for 72 hours with RAE1‐specific siRNA or control siRNA. For overexpression studies, RKO cells stably overexpressing RAE1 and control cells were treated with DMSO or paclitaxel (2 nmol/L) for 6 hours. Cells were fixed as described previously.¹⁹ (link), ²⁰ (link) Immunostaining was performed using the following antibodies at the indicated dilutions: rabbit anti–pericentrin at 1:2000 (Abcam) and mouse anti–α‐tubulin at 1:2000 (Sigma). Fluorescent image acquisition was performed using a fluorescence microscope (BZ‐X700; Keyence) with a 20× objective. Analysis of mitotic defects was performed as described previously.¹⁹ (link) Defects in mitosis were analyzed in all mitotic cells based on five to six photographs, each containing 5‐20 cells, from three independent experiments. Mitotic cells containing more than two spindle poles (determined by tubulin and pericentrin staining) were defined as multipolar.