Fig. 1: MafB undergoes a rapid downregulation in response to LPS and P. aeruginosa, coinciding with the NLRP3 inflammasome priming in macrophages.

a–c BMDMs, PMs and J774A.1 macrophages were treated with or without 100 ng/ml LPS for the indicated time. Levels of the indicated proteins in the cells were determined by western blotting. Vertically stacked bands in (a) originate either from a single membrane or from a replicate membrane, with the same sample loadings. d, e BMDMs and PMs were treated with LPS for the indicated time. mRNA levels of MafB in the cells were determined by real-time PCR. Representative of three independent experiments. f PMs were treated with or without 1 × 10⁷/ml HKPA for 4 h. Protein levels were determined by western blotting. g–i BMDMs, PMs and J774A.1 cells were treated with or without 5 µg/ml CHX for the indicated time. Levels of the indicated proteins were determined by western blotting. j BMDMs were pre-treated with 10 μM MG132 or 10 nM Bafilomycin A1 for 30 min, followed by LPS treatment for 3 h. k J774A.1 cells were pre-treated with or without 1 µM MLN7243 for 30 min, followed by LPS treatment for 3 h. l BMDMs were pre-treated with MG132 for 30 min, followed by treatment of LPS for another 3 h. Cellular extracts were prepared, followed by immunoprecipitation with MafB antibody. Western blotting with the IP and input samples was performed. Mean ± SD; **P < 0.01, ***P < 0.001. Protein densitometric analyses were performed using ImageJ (NIH) and shown under their corresponding bands. Representative of three independent experiments. HKPA heat-killed Pseudomonas aeruginosa, CHX cycloheximide, Bafilo Bafilomycin A1, vehi. vehicle.