Pierce ecl detection reagent

Cells were collected in 600 μL RIPA lysis buffer and stored at −80 °C before protein extraction. Samples were centrifuged at 10 000 g for 10 min at 4 °C and supernatants were harvested. Protein concentrations were measured with Bio‐Rad Protein Assay Dye Reagent Concentrate (500‐0006, Bio‐Rad, Temse, Belgium). Proteins were diluted in RIPA lysis and loading buffers. Heat‐denatured protein samples were separated on 4–12% BisTris‐polyacrylamide gel electrophoresis (NP0322BOX, Invitrogen) followed by transfer to polyvinylidene flouride (PVDF) membranes 0.2 μm (LC2005, Invitrogen). After blocking with 5% (wt/vol) dry milk in TBS containing 0.1% triton (TBST), the membrane was incubated overnight at 4 °C with primary anti‐IRG1 antibody (Ab222411, Abcam) diluted 1 : 250 in 1% (wt/vol) BSA in TBST with constant shaking. After three washing steps with TBST, the membrane was incubated with anti‐rabbit antibody coupled to horseradish peroxidase and revealed by chemoluminescence using Pierce™ ECL detection reagents (Thermo Fisher Scientific). For the second hybridization, the membrane was incubated with anti‐actin antibody (MAB 1501, Millipore, Overijse, Belgium) for 90 min at RT in 1% (wt/vol) BSA in TBST with constant shaking. After three washing steps with TBST, the membrane was incubated with anti‐mouse antibody coupled to horseradish peroxidase and revealed by chemoluminescence.

Western blot was performed as previously described (Tramullas et al., 2010 (link)). Whole-cell lysates were prepared from the lumbosacral region of the spinal cord and total protein was determined using the Quan-iT protein assay kit (Invitrogen, Ireland). Equal amounts of protein were subjected to electrophoresis on 4–12% gradient gels (NuPAGE, Invitrogen, Ireland) and transferred to a polyvinylidene difluoride membrane (Bio Rad, Ireland). Membranes were then incubated with goat anti-GFAP (1:1000; Sigma, Ireland), the astrocyte marker, rabbit anti-CD11b (1:500; Novus Biologicals, UK), a microglia activation marker; and mouse anti β-actin (1:15000; Sigma, Ireland). Immunoreactivity was detected with Pierce ECL detection reagent (Thermo Scientific, IL, USA) and visualized using a luminescent image analyzer (LAS-3000, Fujifilm, Ireland). The relative density of the immunoreactive bands was quantified using Fiji Is Just ImageJ (FIJI) software (Schindelin et al., 2012 (link)) and normalized to the relative density of mouse anti-β actin (Sigma, Ireland) for each sample. No significant differences were observed in the protein expression levels of β-Actin between groups.