Ab73290

Total proteins were extracted with RIPA buffer (Beyotime, Zhejiang, China) containing protease inhibitor cocktail tablets (Roche Applied Science, Mannheim, Germany), and separated by SDS‐PAGE (Invitrogen). Then they were transferred onto PVDF membranes and probed with antibodies against Nav1.5, Kv4.3, and Cav1.2 (ASC‐005, APC‐017, and ACC‐003, Alomone Labs, Jerusalem, Israel), Kir2.1 and Kv1.4 (19965‐1‐AP, and 19697‐1‐AP, ProteinTech Group, Wuhan, Hubei, China), Nup107 and GFP (ab73290, and ab290, Abcam, Cambridge, MA, USA), β‐actin, and GAPDH (66009‐1‐Ig and 60004‐1‐Ig, ProteinTech Group). After washing, blots were treated with the appropriate IRDye 800 conjugated secondary antibody (072‐07‐15‐06 and 072‐07‐18‐06, LI‐COR Biosciences, Lincoln, NE, USA) at room temperature for 1 hours. Images were recorded using the Odyssey infrared imaging system and analysed using the Odyssey Application Software v2 (LI‐COR Biosciences).

The RIP experiment was performed using a Millipore EZ‐Magna RIP RNA‐Binding Protein Immunoprecipitation kit (17‐701, Millipore, Bedford, MA, USA) according to the manufacturer's instructions. NRVMs were infected with Ad‐Nup107 or Ad‐GFP for 48 hours and harvested for the endogenous Scn5a mRNA immunoprecipitation. Antibodies used for RIP included rabbit polyclonal IgG (Millipore, PP64), GFP, and Nup107 (ab290 and ab73290, Abcam), and 5 μg of antibody was used per RIP reaction. The immunoprecipitated RNA was extracted with Trizol and reverse‐transcribed with PrimeScript RT reagent Kit (Takara). All RIP assays were performed with biological duplicates. The RIP‐PCR was used to measure the amount of Scn5a mRNA in pull‐down samples. The data were demonstrated as the percentage of input GAPDH mRNA of its respective group. P values were obtained using two‐tailed Student's t‐test.