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. Author manuscript; available in PMC: 2018 Nov 1.
Published in final edited form as: J Immunol. 2017 Sep 20;199(9):3249–3260. doi: 10.4049/jimmunol.1700823

Figure 2. p205 knockdown using shRNA results in impaired inflammasomes activation.

Figure 2

BMDM transduced with shRNA targeting either (A) 3′UTR or (B) CDS of p205 gene were inspected for expression of p205, Mnda and p204 mRNA relative to β-actin mRNA and normalized to expression in GFP shRNA BMDM. (C) Heatmap of PYHIN gene expression in p205 KD 3′UTR and KD CDS BMDM compared to control (Ctl) BMDM. The p205 knockdown BMDMs were primed with LPS (200ng/ml) for 3h and then stimulated with transfected pdAdT (1μg/ml for 6h), transfected ISD (3μM for 6h), Nigericin (10μM for 1h), ATP (5μM) or, stimulated alone with Sendai virus (SV; overnight) or poly I:C (overnight). Secreted (D) IFNβ and (E) IL-1β levels were assessed by ELISA. (F) GFP shRNA CTL, p205 KD 3′UTR and KD CDS were primed with LPS (200ng/ml) for 3h and then stimulated with pdAdT (1 μg/ml for 6h) or Nigericin (10 μM for 1h) and the supernatants and the lysates from the macrophages were immunoblotted for pro-IL1β (35 kD), cleaved form of IL-1β (p17), pro-caspase 1 (45kD) and the active subunit of caspase 1(p20).