Mouse hsp70

Cell lysis, SDS-PAGE, and Western blotting were as previously described (Hames et al., 2005 (link)). For Western blotting, primary antibodies used were 1 µg/ml rabbit Nek6 (O’Regan and Fry, 2009 (link)), 1 µg/ml rabbit Nek7 (O’Regan and Fry, 2009 (link)), 0.3 µg/ml mouse α-tubulin (Sigma-Aldrich), 0.5 µg/ml mouse Flag (Sigma-Aldrich), 0.5 µg/ml mouse Hsp70 (Santa Cruz Biotechnology, Inc.), 0.5 µg/ml mouse Hsp72 (Enzo Life Sciences), 0.5 µg/ml mouse Hsc70 (Santa Cruz Biotechnology, Inc.), 1 µg/ml rabbit pHsp70 (this study), 0.8 µg/ml goat Nek9 (Santa Cruz Biotechnology, Inc.), 1 µg/ml rabbit GAPDH (Cell Signaling Technology), 1 µg/ml mouse phospho–histone H3 (EMD Millipore), 0.2 µg/ml rabbit γ-tubulin (Sigma-Aldrich), 0.4 µg/ml mouse TACC3 (Abcam), 0.5 µg/ml rabbit ch-TOG (Bethyl Laboratories, Inc.), and 1 µg/ml mouse Cep55 (Santa Cruz Biotechnology, Inc.). Nek7 antibodies were raised against a peptide corresponding to amino acid residues 12–28 conjugated to keyhole limpet hemocyanin via an N-terminal cysteine (C-OVPQFQPQKALRPDM). Secondary antibodies were HRP-labeled anti–mouse, anti–rabbit, or anti–goat IgGs (1:1,000; Sigma-Aldrich) or alkaline phosphatase–conjugated IgGs (1:7,500; Promega). HRP-labeled blots were detected by enhanced chemiluminescence (Thermo Fisher Scientific).