Immunoblotting Analysis of BMDM Cells

Immunoblottings for BMDMs were performed as previously described by Tarrago et al. (23 (link)). Cells were homogenized and lysed in NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40) supplemented with 50 mM β-glycerophosphate, 5 mM NaF and a protease inhibitor cocktail (Roche). For PARylation analysis 100 μM tannic acid was included in the lysis buffer. After 20 minutes of incubation on ice, the samples were centrifuged at 12,000 rpm for 10 minutes at 4°C. Protein concentrations in the supernatants were determined by Bio-Rad protein assay. Lysates were separated by SDS–PAGE, and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). Enhanced chemiluminescence detection was performed using SuperSignal West Pico or Femto Chemiluminescence Substrate (Thermo Scientific). Films were scanned and densitometry was performed using ImageJ. The following antibodies and their dilutions were used for immunoblotting: anti-mouse pp65 (S536) (Cell Signaling Technology; 3033, 1:1,000) anti-mouse p65 (Cell Signaling Technology; 8242, 1:1,000), actin (Cell Signaling Technology; 8457, 1:5,000), PAR (Trevigen, 4335, 1:1,000).