Anti vrk2

Cells were washed once in PBS and then lysed in 141 mM Tris Base, 106 mM Tris HCl, 2% LDS, 1% Benzonase nuclease (E1014, Millipore), pH 8.5, at room temperature for 15 min and snap frozen in liquid nitrogen. The protein concentration was determined using Rapid Gold BCA Protein Assay Kit (Pierce). Cell lysates (20 µg) were run on NuPAGE 4-12% Bis-Tris protein gels (Invitrogen) using standard procedures, transferred to 0.2 µm polyvinylidene fluoride membranes and blocked in 5% milk in TBST (0.05% Tween 20 in Tris-buffered saline) for 1 h at room temperature before overnight incubation with primary antibodies. Membranes were washed thrice in TBST then incubated with a HRP conjugated secondary antibody for 1 h at room temperature in TBST with 5% milk, and signals were detected by chemiluminescence (Clarity ECL, Biorad). Antibodies used in immunoblotting were as follows: rabbit polyclonal anti-Vinculin (Cell Signaling Technology, #13901), anti-Haspin (Abcam, ab21686, lot 156753), and anti-H3T3ph (B8634; (9)) with anti-rabbit IgG HRP (Cell Signaling Technology, #7074) and mouse monoclonal anti-VRK1 (Abcam, ab171933), anti-VRK2 (Santa Cruz, sc-365199), anti-H3S10ph (Millipore, 05-806), anti--tubulin (B-5-1-2, Sigma, T5168) and anti-MPM2 (Millipore, 05-368) with anti-mouse IgG HRP (Cell Signaling Technology, #7076).