Lf pvdf

Total proteins were extracted using the Protein Extraction Reagent Type 4 (P4, Sigma‐Aldrich). Cells were sonicated with 10 pulses of 15 s at 45 W Labsonic 1510 Sonicator (Braun, Melsungen, Germany) and clarified by centrifugation for 10 min at 10 000 g. Protein concentration was determined by the Bio‐Rad Protein Assay, based on Bradford's method. Twenty micrograms of proteins were separated by SDS/PAGE (Novex Tris‐Glycine gels) according to the Laemmli protocol [22 (link)] and then transferred to nitrocellulose (0.22 μm; Bio‐Rad) or LF PVDF (0.45 μm; Bio‐Rad) by wet transfer and Towbin blotting buffer (50 mm Tris, 150 mm NaCl, 20% v/v methanol). Membranes were probed with the primary antibodies diluted in 5% w/v nonfat dry milk or 5% BSA in TBS‐T. The primary antibodies used in this study were anti‐HDAC2 (CST) and anti‐Lamin A/C (CST). The utilized secondary antibodies were Alexa Fluor 790 (Thermo Fisher Scientific). Immunoreactive bands were recorded using the enhanced chemiluminescence (Advansta, Menlo Park, CA, USA) or fluorescence acquisition by ChemiDoc Touch Imaging System (Bio‐Rad). The whole lane normalization (WLN) strategy was adopted in all western blot analyses using a trihalo compound for protein visualization [23 (link), 24 (link), 25 (link)]. Acquired images were analyzed by image lab software 5.2.1 (Bio‐Rad) [26 (link)].

Na_v1.4 cDNA plasmids were transfected into HEK293 cells 12–18 hr after plating at 70% confluency using standard calcium-phoshate reagents. 18–24 hr post transfection the HEK293 cells were washed twice with PBS and scraped in PBS supplemented with PMSF (0.1 mM final concentration)and Benzamidine (0.75 µM final concentration) and spun at 4000 g. The cell pellet was resuspended in solubilization buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% Triton, 0.5 µg/ml Pepstatin, 2.5 µg/ml Aprotinin, 2.5 µg/ml Leupeptin, 0.1 mM PMSF, 0.75 mM Benzamidine) on ice and Vortex Genied for 15mins at 4 C. Non-solubilized protein was removed by centrifugation at 20,000 g. Equal cell-lysate was loaded on a 3-15% gradient SDS-page in the presence of 1% 2 mercaptoethanol, separated at 55 V O/N and transferred to 0.45 µM LF PVDF (Bio-Rad, CA, USA). PVDF was immunoblotted using anti-Human influenza hemagglutinin (HA) antibody (1:2000; Biolegend, CA) in 2% NF milk and imaged on LI-COR Odyssey Imaging System (LI-COR, NE, USA).