Mouse anti connexin 32 monoclonal antibody

Western blot analysis was performed as described previously (Arnoldussen et al. 2015 (link)). Briefly, concentrations of the extracted protein were measured using NanoDrop-8000 (Thermo Scientific). 100 μg of protein for each sample was resolved on AnykD Mini protean TGX stain free gels (Bio-Rad) and transferred to a PVDF membrane (Transblot Turbo Transfer pack, Bio-Rad). The Trans-Blot Turbo blotting system (Bio-Rad) was used for transfer. Antibodies used were as follows; Gja1 (rabbit connexin 43 polyclonal antibody, Cell Signaling Technology), Gjb1 (mouse anti-connexin-32 monoclonal antibody, Millipore), Gjb2 (goat anti-Gjb2 polyclonal antibody, Abcam), Sodium Potassium ATPase alpha 1 (mouse anti-NaK ATPase α1 monoclonal antibody, Abcam) and α-Tubulin (rabbit monoclonal antibody, Cell Signaling Technology). Horseradish peroxidase HRP-conjugated secondary antibodies against rabbit and mouse (both from Cell Signaling Technology) and against goat (Santa Cruz Biotechnology, INC) were used.

IL1-WT and IL1-KO cells were cultured on cover slips and were allowed to attach for 24 h before exposure to 5 μg/ml of CNT-1 and CNT-2 for 24 h. Cells were then fixed for 20 min in 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 in PBS for 5 min. Cover slips were blocked by incubation with 5 % BSA in 0.1 % PBS-Triton X-100 for 1 h at room temperature and incubated overnight with a primary antibody against Gja1 (rabbit connexin 43 polyclonal antibody, Cell Signaling Technology), Gjb1 (mouse anti-connexin-32 monoclonal antibody, Millipore) or Gjb2 (goat anti-Gjb2 polyclonal antibody, Abcam) in 3 % BSA in PBS at 4 °C in a humidified chamber. Secondary antibodies anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 488 or anti-goat Alexa Fluor 488 (all from Molecular Probes) were left on the cells for 1 h at room temperature in a humidified chamber. To visualize cell nuclei cells were counterstained with Hoechst (Sigma-Aldrich). Mowiol was used for mounting of the cover slips. Fluorescence was observed using a laser scanning microscope (LSM 710, Zeiss) and pictures were taken with an AxioCam camera (Zeiss).