Odyssey western blotting kit

Western blotting was performed by loading 30 μg protein on 10% (w/v) tris-glycine denaturing gels and separating the proteins by electrophoresis, then transferring them to a PVDF membrane. After blocking, the membrane was incubated with primary antibodies, anti-PARP rabbit monoclonal antibody (1:1000, Cell Signaling Technology, Invitrogen Fisher Scientific, Illkirch, France), anti-caspase-3 rabbit monoclonal antibody (1:1000, Cell Signaling Technology), anti-Bax rabbit monoclonal antibody (1:1000, Cell Signaling Technology), anti-Bcl-2 rabbit monoclonal antibody (1:1000, Cell Signaling Technology) at +4 °C overnight. Following incubation, the membrane was incubated with anti-β-actin mouse monoclonal antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. After washing, the membrane was incubated with infrared labeled secondary antibodies (Odyssey Western Blotting kit, LI-COR Biosciences, Lincoln, NE, USA) at room temperature for 1h. Proteins were visualized, and quantified by scanning the membrane on an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) with both 700- and 800-nm channels.

The ganglia were mechanically dissociated using Precellys 24 Bertin technologies into a RIPA lysis buffer (Thermofisher, Rockford, IL, USA). After tissue lysis, homogenates were centrifuged (4 °C, 10,000 rpm) for 10 min. Pellets were resuspended in a Laemmli buffer (Bio-Rad). After protein quantification, 50 µg of protein was used to perform electrophoresis (Tris-HCl PAGE-Gel (Bio-Rad laboratories Inc., Hercules, CA, USA). The transfer of proteins was performed using an activated-methanol membrane (0.35 A) (Merck Millipore, Tullagreen, Carrigtwohill, Co, Cork, Ireland, Immobilon FL Transfer Membrane) at 4 °C for 90 min. Blocking and antibody incubation were performed using the IBindTM Flex Western System SLF2000 (Invitrogen). The intensity of each band was determined by pixel density integration using LI-COR^® Odyssey Western Blotting Kits. Antibodies used are presented in Table 1.

At 24hpi, culture medium was removed and cells were resuspended into RIPA lysis buffer (Thermofisher, Rockford, IL, USA). After cell lysis, homogenates were centrifuged at 10,000 rpm for 10 min at 4 °C. Cell lysates were resuspended into Laemmli buffer (Bio-Rad Laboratories Inc, Hercules, CA, USA). After protein quantification, 50 µg of protein was used to perform electrophoresis (Tris-HCl PAGE-Gel, Bio-Rad laboratories Inc, Hercules, CA, USA). Transfer of proteins was performed using activated-methanol membrane (Immobilon FL Transfer Membrane, Merck Millipore, Tullagreen, Carrigtwohill, County Cork, Ireland) at 4 °C during 1h30 with 0.35A. Blocking and antibody incubation (shown in Table 2) were performed using the IBind^TM Flex Western System SFL2000 (Thermofisher, Invitrogen, Carlsbad, CA, USA). The intensity of each band was determined by pixel density integration using LI-COR^® Odyssey Western Blotting Kits.