Rabbit anti his

For immunofluorescence, transfected cells were fixed with 100% methanol for 10 min at room temperature (RT). Cells were blocked in Phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) for 1h at RT. Next, the primary antibody dilution containing rabbit anti-His (1:1000) (Bethyl Laboratories Inc.) in PBS with 0.1% BSA was applied for 1h at RT. Cells were then incubated with 2ug/mL of secondary antibody dilution containing Alexa Fluor 568 goat anti-mouse (Thermo Fisher Scientific, Rockford, IL, USA) in PBS with 0.1% BSA for 1h at RT. Lastly, cells were washed in PBS and mounted with Fluoroshield^TM (Sigma-Aldrich).
For imaging of fluorescently tagged proteins, transfected cells were fixed with 4% paraformaldehyde for 30 min at RT and mounted with Fluoroshield^TM (Sigma-Aldrich). Images were taken with a Zeiss LSM 700 confocal microscope using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective. Zeiss ZEN imaging software was used to control all imaging parameters.

For western blot, cell protein lysates were prepared 48 h after transfection. Proteins were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 150 V for 1.5 h. The gel was transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad Inc., Mississauga, ON, Canada) at 1.3 A and 2.5 V for 7 min. The membrane was washed in PBS buffer and blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature (RT). The membrane was then incubated with the primary antibody solution overnight at 4 ºC. The following primary antibodies were used for the western blot: rabbit anti-HIS (Bethyl Laboratories Inc., Montgomery, TX, USA) at 1:1000, mouse anti-HA (Roche Holding AG, Basel, Switzerland) at 1:500, mouse anti-β-actin (Sigma-Aldrich Chemie GmbH, Munich, Germany) at 1:1500 concentrations. The secondary antibodies, anti-mouse iRDye 800 and anti-rabbit iRDye 680 (LI-COR Biosciences, Lincoln, NE, USA), were used at 1:15,000 concentration. Imaging was performed using the Odyssey^® CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Transfected cells were fixed with 4% paraformaldehyde for 20 min at RT, washed with PBS, and mounted with ProLong Antifade Mountant (Thermo Fisher Scientific, Rockford, IL, USA) for imaging. Samples were visualized using a Zeiss LSM 700 confocal microscope using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective. Zeiss ZEN 2010 program was used to control imaging specifications. The gap junction area was determined using ImageJ by tracing the gap junction area with a free hand tool followed by quantification using the measure tool. ImageJ software was also used to analyze co-localization data.
In the case of immunofluorescence, transfected cells were fixed with ice-cold 100% methanol for 10 min at RT. Cells were blocked using PBS supplemented with 2% BSA for 1 h at RT. Primary antibody, rabbit anti-HIS (Bethyl Laboratories Inc., Montgomery, TX, USA) at 1:1000 concentration, was diluted in PBS with 0.1% BSA and applied to cells for 1 h at RT. Cells were then incubated in the secondary antibody solution containing 2 μg/mL of Alexa Fluor 568 goat anti-mouse (Thermo Fisher Scientific, Rockford, IL, USA) and 2 μg/mL of Alexa Fluor 488 donkey anti-rabbit (Thermo Fisher Scientific, Rockford, IL, USA) in PBS with 0.1% BSA for 1 h at RT. Cells were washed in PBS and mounted with FluoroshieldTm (Sigma-Aldrich Chemie GmbH, Munich, Germany).