Phospho aurora a

Lysate samples containing 200–600 μg cell were precleared using 25 μl protein G magnetic beads (New England BioLabs, Frankfurt, Germany); after 1‐h incubation and removal of the beads, the precleared lysate was incubated with 3 μg mAb33 anti‐K_V10.1 antibody for 1 h, and 25 μl clean protein G magnetic beads were added and incubated for 1 h. The recovered beads were then washed with 50 mM Tris–HCl pH 7.4, 300 mM NaCl, 5 mM EDTA, and 0.1% Triton X‐100. Bound proteins were eluted with PAGE loading buffer. Samples were separated in 3–8% or 4–12% gradient polyacrylamide precast gradient gels (Thermo Fisher Scientific), transferred to nitrocellulose membranes, and immunoblotted with polyclonal anti‐K_V10.1 antibody (9391).
For Western blot of additional proteins, the following antibodies were used: pan‐14‐3‐3 (sc‐629, Santa Cruz Biotechnology, Santa Cruz, CA), Actin (I19, Santa Cruz), phospho‐Aurora A (#3079, Cell Signaling Technologies, Danvers, MA), Aurora A (#12100; Cell Signaling), Calnexin (ADI SPA 860, Enzo Life Sciences, Lörrach, Germany), CortActin (ab81208, Abcam), phospho‐cortActin (Y466, SAB4504373, and Y421, SAB4504372, both from Sigma), γ‐tubulin (sc7396, Santa Cruz), and human transferrin receptor (612125, BD).

Cells were lysed with M‐PER™ Mammalian Protein Extraction Reagent (Catalog #78503; Thermo Fisher) on ice for 30 min with vortexing and then sonicated with Q125 Sonicator from Qsonica (Newtown, CT, USA). Lysates were centrifuged at 18,407  g for 15  min at 4°C, and the protein concentration was measured using a BCA kit (Catalog #23225; Thermo Scientific). The cell lysates were adjusted to 5 mg mL⁻¹ to run on SDS‐PAGE. Following electrophoresis, proteins were transferred onto PVDF membranes and incubated with rabbit monoclonal antibodies against Aurora‐A, Phospho‐Aurora‐A, PLK1, Phospho‐PLK1 (Thr210), mTOR or Phospho‐mTOR (Ser2448) from Cell Signaling Technology (Danvers, MA, USA), or with monoclonal antibodies against 4E‐BP1 or Phospho‐4E‐BP1 from Santa Cruz Biotechnology (Dallas, TX, USA). ECL kit (Catalog #1705061) and the Gel Doc™ XR+ System from Bio‐Rad (Hercules, CA, USA) were used to develop and acquire Western blot images. Band intensity was analysed using the ImageJ software (NIH) and normalised by loading control β‐actin levels.