Rabbit anti mouse igg hrp

Western blot analysis was performed on fully-confluent Cre-responsive cell lines harvested from 24-well plates 6 or 22 hours after the single administration of PTD-Cre (15 nM) or syn-mRNA-Cre (2.1 nM). The cells were lysed using RIPA buffer composed of 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0 [27 (link)]. Cell lysates (14 μg total protein per well) were mixed with 4x Laemmli loading buffer containing 8% SDS, 40% glycerol, 0.02% bromophenol blue, 250 mM Tris, and 20% 2-mercaptoethanol (all from Sigma-Aldrich), pH 6.8, heated at 95°C for 3 min, and run on a 15% polyacrylamide gel and transferred to PVDF membranes (Merck Millipore) using a Pierce G2 electroblotter (Thermo Fisher Scientific). The membranes were blocked with 3% BSA (Sigma-Aldrich). Primary antibodies included a rabbit anti-T7 antibody (Abcam, Cambridge, UK) for detecting Cre recombinase (1:2000 dilution) and a mouse anti-beta-actin antibody (Sigma-Aldrich) as a loading control (1:7500 dilution). The secondary antibodies included goat anti-rabbit IgG-HRP (Merck Millipore) and rabbit anti-Mouse IgG-HRP (Thermo Fisher Scientific), each diluted 1:50000. Chemiluminescent SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used for detection. The signals were acquired using a G:BOX Chemi XR5 (Syngene, Cambridge, UK).

HEK293T cells that heterologously expressed WT or mutant Na_V1.5 channels were lysed and samples of equal total protein amount (standard BCA-assay) were run on 10% SDS-PAGE. The electrophoresed proteins were transferred to nitrocellulose-membrane (#RPN303D, Amersham Hybond ECL, GE Healthcare Europe). Subsequently, the membrane was cut between the 75 kDa and 50 kDa band (Precision Plus Protein dual color marker #161-0374, BIO-RAD). The section with proteins of higher molecular weight (≥75 kDa) was incubated with a primary rabbit anti-Na_V1.5 antibody (ASC-005, alomone Labs, 1:1000 in 1x TBST with 1% BSA), whereas the other membrane section (≤50 kDa) received a mouse anti-GAPDH antibody (A-3, sc-137179, Santa Cruz Biotechnology, 1:4000 in 1x TBST with 1% BSA). Goat anti-rabbit IgG HRP (#31463, Thermo Fisher, 1:20000 in 1xTBST with 1% BSA) or rabbit anti-mouse IgG HRP (#31455, Thermo Fisher; 1:25000 in 1x TBST with 1% BSA) were used as secondary antibody to visualize Na_V1.5 and GAPDH by Super Signal^TM West Pico/Femto Luminol (Thermo Fisher) and Fusion imaging system (Vilber Lourmat).

Cells were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, Mass) with 1% proteinase inhibitors (Roche Diagnostics). Lysates were boiled in the presence of 2 X Laemmli Buffer. Protein concentrations were measured using Pierce BCA Protein Assay (Thermo Fisher Scientific, UK). A total of 20 μg total protein was subjected to SDS-PAGE analysis and electro transferred onto polyvinylidene difluoride membranes (Millipore, Temecula, Calif). Membranes were blocked with milk, probed with primary and secondary antibodies, and visualized with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). The following antibodies were used: ADA2 (ab154619, Abcam), ACTN (MAB 1501R; Merck Millipore, Burlington, Mass), Goat anti-Rabbit IgG, HRP (ThermoFisher) and Rabbit anti-mouse IgG, HRP (ThermoFisher).