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Fig. 2 | Respiratory Research

Fig. 2

From: Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination

Fig. 2

CSE triggers NLRP3 protein degradation via the ubiquitin proteasome system. a Above: Overall abundance of ubiquitin (arrows) increases with CSE exposure. THP1 cells were incubated with the indicated concentrations of CSE for 20 h. Below: Protein levels of ubiquitin, NLRP3, NLRP6, GAPDH, and β-actin were determined by immunoblotting. Membranes were stripped to detect multiple proteins. Ubiquitin and GAPDH are from the same blot, and NLRP3, NLRP6, and β-actin are from a separate blot. b THP1 cells were incubated with or without CSE at 80 μg/mL for 17 h. Cells were lysed and immunoblotted for NLRP3 protein (left panel, input; cell lysates). In the right panel, ubiquitin was immunoprecipitated, followed by NLRP3 immunoblotting. Modestly increased intensity of several bands (arrows) with CSE exposure are observed, and corrected for input the results are shown graphically (bottom). The bar graph represents mean ± S.D. from two independent experiments. c THP1 cells were incubated with or without CSE at 80 μg/mL, and with or without MG-132 at 20 μM or leupeptin at 40 μM for 16 h. As MG-132 is water insoluble, dimethyl sulfoxide (DMSO) was used as a vehicle. Control was normalized by the amount of DMSO used (0.1%). Immunoblotting was performed to determine NLRP3, NLRP6, and β-actin protein levels. The blots shown are representative of two to three independent experiments

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