Fig 4.

Roles of reactive oxygen species and proteasome activity in Nlrp1b activation. (A) Plasmids pcDNA3–procaspase-1-T7, pcDNA3–pro-IL-1β–HA, and pNTAP-Nlrp1b were transfected into HT1080 cells. Approximately 24 h after transfection, cells were left untreated or were treated with LeTx, 50 mM 2DG, and 10 mM NaN₃ or 50 mM 2DG in the absence or presence of 25 mM N-acetyl cysteine (NAC). After 3 h, cell lysates were collected and probed for HA-tagged pro-IL-1β and β-actin by immunoblotting; supernatants were immunoprecipitated with anti-HA antibodies and probed for HA-tagged pro-IL-1β and IL-1β by immunoblotting. (B) Cell lysates from panel A were assayed for ATP. (C) Cells were transfected with pcDNA3–procaspase-1-T7, pcDNA3–pro-IL-1β–HA, and pNTAP-Nlrp1b. Approximately 24 h after transfection, cells were treated with LeTx or 50 mM 2DG and 10 mM NaN₃ in the presence or absence of 10 μM MG-132. After 3 h, cell lysates were collected and probed for HA-tagged pro-IL-1β; HA-tagged pro-IL-1β and IL-1β in supernatants were detected as described above. (D) Cell lysates from panel C were assayed for ATP. Blots shown represent at least three independent experiments. The asterisk indicates an ∼25-kDa HA-tagged pro-IL-1β-cleaved product. Error bars represent standard deviations for at least three independent experiments.