Anti cenp c

Western blot was performed as described previously (Hao et al., 2020 (link)). Cells were collected and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% Tween-20, 0.5% NP-40, 2 mM PMSF, 2.5 mM β-glycerophosphate (all from Sigma-Aldrich) and protease inhibitor cocktail tablet (complete Mini, Roche Diagnostics). Equal amounts of protein were resolved by 10% Tris-Glycine gel (SDS-PAGE). Proteins were blotted onto a nitrocellulose membrane, blocked with blocking buffer (1xTBS, 0.1% Tween-20) with 5% w/v nonfat dry milk and incubated with the corresponding primary/secondary antibodies: anti-CENP-C (1:1000, Abcam), anti-GFP (1:1000, BioVision), anti-Actin (1:5000, Abcam). After incubation with peroxidase conjugated secondary antibodies (1:5000, Abcam), blots were developed with Supersignal West Pico Chemiluminescent Substrate (Pierce) and exposed to film (SAGECREATION, MiNiChemi).

IF on settled interphase cells and metaphase spreads were performed as previously described (Chen et al., 2014 (link)). Primary antibodies: anti-CENP-A (chicken, 1:1,500; Blower and Karpen, 2001 (link)) or anti-CID (rabbit, 1:500; Abcam), anti-CENP-C (guinea pig, 1:500; Erhardt et al., 2008 (link)), anti-Ndc80 (chicken, 1:200; Cane et al., 2013 (link)), anti-GFP Alexa 488-conjugated (rabbit, 1:100; Invitrogen), or anti-GFP (chicken, 1:500; Abcam), and anti-HA (mouse, 1:500; Covance).
For salt extractions, settled cells were incubated with PBS-D (0.1% digitonin) with or without 0.5 M NaCl for 30 min (Perpelescu et al., 2009 (link)) before 37% formaldehyde was added to the solution to a final concentration of 3.7% followed by 10 min of incubation before proceeding with IF.

RIP-seq was carried out as previously described (Zhang et al., 2013 (link)) with modifications. HeLa cells (1 x 10⁷) were harvested, resuspended in lysis buffer (25 mM Tris pH7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol, 1 mM PMSF, and 2 mM VRC, with Protease Inhibitor Cocktail, Roche) and then lysed on ice for 30 minutes. After incubation with anti-CENP-C (2 μg, Abcam) or anti-IgG (Santa Cruz) antibodies, the beads were washed once in lysis buffer and four times in washing buffer (50 mM Tris pH7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40), followed by elution with extraction buffer C (100 mM Tris pH6.8, 4% SDS, 12% β-mercaptoethanol, 20% glycerol) at room temperature for 10 minutes. One-third of the RIP materials was used for immunoblotting and the other two-thirds were used for RNA extraction. Each RNA sample was treated with DNase I (Ambion, DNA-freeTM kit). We adhered to the protocols supplied with NEB Next® UltraTM Directional RNA Library Prep Kit for Illumina®.