Cells were lysed with RIPA buffer (25 mM Tris-Cl pH 7.6, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 1% sodium deoxycholate, 1% PMSF, and 1% proteinase inhibitor cocktail; Roche) and samples were quantified using a protein assay (Micro BCA; ThermoFisher Scientific). The same amount of protein was separated using SDS-PAGE (10%) and the gel scanned with a Typhoon 9410 Gel and Blot Imager (GE healthcare life sciences).
For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451). Blots were visualized using secondary HRP-conjugated anti-rabbit or anti-mouse antibodies and SuperSignal West Pico or Femto PLUS Chemiluminescent Substrate (ThermoFisher Scientific). The data was analyzed with ImageJ.
For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451). Blots were visualized using secondary HRP-conjugated anti-rabbit or anti-mouse antibodies and SuperSignal West Pico or Femto PLUS Chemiluminescent Substrate (ThermoFisher Scientific). The data was analyzed with ImageJ.