Journal: Respiratory Research

Article Title: Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination

doi: 10.1186/s12931-016-0485-6

Figure Lengend Snippet: CSE decreases the half-life of the NLRP3 protein. a THP1 cells were incubated with or without CSE at 80 μg/mL for 18 h prior to CHX exposure at 40 μg/mL for the indicated time periods. Cells were collected and assayed for NLRP3 and β-actin (loading control) by immunoblotting for a half-life study. Representative images are shown. b Densitometric plots of NLRP3 protein decay versus time of CHX exposure with best fit lines, depicting the pooled data mean ± S.D. of three independent experiments. c Primary human macrophages from peripheral blood were transfected with 4 μg of either WT NLRP3 -V5 or the point mutant K689R NLRP3 -V5 plasmid. Following transfection, the cells were incubated with or without CSE at 120 μg/mL for 21 h, prior to CHX exposure at 40 μg/mL at different time points for a half-life study. Cells were collected and assayed for NLRP3-V5 and β-actin by immunoblotting. d Densitometric plots of adjusted NLRP3 protein decay over time under different conditions were best fitted. The half-life of WT NLRP3 protein was reduced with CSE exposure, while a K689R NLRP3 mutant was not. Data are mean ± S.D. of two independent experiments

Article Snippet: Human peripheral blood macrophages and human macrophage cell culture medium were purchased from Celprogen (Torrance, CA).

Techniques: Incubation, Control, Western Blot, Transfection, Mutagenesis, Plasmid Preparation

Journal: Respiratory Research

Article Title: Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination

doi: 10.1186/s12931-016-0485-6

Figure Lengend Snippet: CSE reduces LPS-induced NLRP3 abundance and release of cytokines. a THP1 cells were incubated with or without 80 μg/mL of CSE, with or without 400 ng/mL of LPS, for 18 h (four groups; none, CSE, LPS, and LPS + CSE). Human primary macrophages derived from monocytes were incubated with or without 120 μg/mL of CSE, with or without 200 ng/mL of LPS, for 20 h. Protein levels of NLRP3, NLRP6, β-actin or GPADH were determined by immunoblotting. Culture medium (supernatants) were also collected for immunoblotting of NLRP3 and NLRP6. The relative densitometries of NLRP3 protein adjusted for loading control (β-actin or GPADH) are shown in the bottom panels. Data are mean ± S.D. of two independent experiments. b Equal numbers of THP1 (3 × 10 6 cells) were plated in equivalent amounts of culture medium and incubated with or without 80 μg/mL of CSE, with or without 400 ng/mL of LPS, for 18 h. Cells were then exposed to 5 mM of ATP for 15 min. Culture medium was precipitated with TCA for immunoblotting of IL-1β, IL-18, and caspase-1. Also, equal numbers of human monocyte-derived macrophages in equal amounts of culture medium were incubated with or without 120 μg/mL of CSE, with or without LPS at 400 ng/mL, for 40 h. Cells were then exposed to 5 mM of ATP for 30 min. Cell lysates and culture medium were collected for immunoblotting. p45 and p20 are probed in the same blot, but presented in different exposure. IL-1β and IL-18 are from the same blot

Article Snippet: Human peripheral blood macrophages and human macrophage cell culture medium were purchased from Celprogen (Torrance, CA).

Techniques: Incubation, Derivative Assay, Western Blot, Control