Anti nek2

Total lysate protein samples from the ZG and ZF fractions (30 µg from each) were sorted in 10% SDS-PAGE. To test the Nek2 protein isoforms, we used a commercial HeLa nuclear extract (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for positive control of the antibody. We also obtained Y1 and primary rat adrenal cell culture total protein extracts, as described in [9] (link). After electrophoresis, the gel was electroblotted onto Hybond-C nitrocellulose membranes using a semi-dry Bio-Rad apparatus. Non-specific sites were blocked overnight at 4°C with 5% dried non-fat milk dissolved in TBS (150 mM NaCl, 10 mM Tris, 1% Tween 20, pH 7.5). The membranes were subsequently incubated with anti-Nek2 (1∶1000; Santa Cruz) or β-actin (1∶2000, Santa Cruz) for two hours at room temperature. Proteins were detected using chemiluminescent secondary peroxidase-conjugated anti-rabbit or anti-mouse polyclonal antibodies (ECL-Amersham-Pharmacia, Piscataway, NJ, USA). The immunoblot results were quantified by densitometry using the Gel-Pro Imager and Gel-Pro Imager kit Version 1.0 quantification program for Windows. The average and standard error were calculated for the data obtained from each fraction. Ponceau staining was used to monitor protein transfer amounts and total protein loaded.

Protein complexes were collected by immunoprecipitation. Briefly, affinity purified antibody to PPP1R42 was incubated with precleared cell lysate (1 mg protein) followed by anti-rabbit IgG beads. After transfer to membrane, immunoprecipated proteins were detected with anti-Nek2 (1:500; sc-33,167; Santa Cruz Biotechnology; Dallas, TX) or anti-AurA antibodies (1:500; PC742; EMD Millipore, Billerica, MA), and Veriblot anti-rabbit HRP (Abcam; Cambridge, MA). Use of the Veriblot secondary prevents detection of IgG heavy chain. Negative control for coimmunoprecipitation was precleared lysate incubated with no antibody.

Total cell extracts were obtained by lysis in RIPA buffer as described [28 (link)]. Cellular fractionation was performed as described in [40 (link)]. Western Blot were performed as described [26 (link), 28 (link)] using following primary antibodies: mouse anti-hnRNP F/H (dilution 1:1000, Abcam), anti-VIMENTIN (dilution 1:2000, Sigma Aldrich), anti-TUBULIN (dilution 1:1000, Sigma Aldrich), anti-NEK2 (dilution 1:500, Santa Cruz), anti-NF-YA, anti-HSP90a/b, anti-GAPDH, anti-SRSF1 (dilution 1:1000, Santa Cruz); goat anti-MATRIN3 (1:500, Santa Cruz); rabbit anti-H3 (dilution 1:1000, Abcam) and anti-E-CADHERIN (1:1000, Cell Signalling). Densitometric analysis were performed using Alliance system software (UVITEC, Cambridge).