Figure 4. RNH1 increases caspase-1 degradation through the proteasome.

(A) Total cell lysates from WT and RNH1-KO THP1 cells were analysed for pro-caspase-1 and ASC expression by Western blot. Blots are representative of two independent experiments. (B) qRT-PCR analysis for CASP-1 and ASC mRNAs from WT and RNH1-KO THP1 cells. mRNA levels are normalized to 18S rRNA expression. Data shown as mean ± SEM from three independent experiments. (C, D, E) WT and RNH1-KO THP1 cells were treated with actinomycin D or cycloheximide or with the proteasome inhibitor MG-132 for indicated time duration. Cell lysates were analysed for pro-caspase-1 protein levels by Western blot. Blots are representative of three independent experiments. (F) HEK293T cells were treated with or without MG-132 and transfected with Flag-tagged caspase-1 with different concentration of GFP-tagged RNH1 plasmid as indicated. Cells were harvested and cell lysates were analysed for caspase-1 by Western blot with the indicated antibodies. Blots are representative of three independent experiments. (G) HEK293T cells were transfected with Flag-tagged OMI/HTRA2 plasmid with different concentrations of GFP-tagged RNH1 plasmid as indicated. Cells were harvested and cell lysates were analysed by Western blot with indicated antibodies. Blots are representative of three independent experiments. CASP-1 (caspase-1), ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain), HTRA2 (High temperature requirement protein A2), and GFP (green fluorescent protein).

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