Skip to main content
. 2019 Apr 8;9:5788. doi: 10.1038/s41598-019-42298-4

Figure 1.

Figure 1

Caspase-1 induces the release of EEA1. (A) Canonical NLRP3 activation by hypotonic solution (90 mOsm, 1 h) in LPS-primed THP-1. Cell lysate and concentrated cell-free supernatant were analyzed by immunoblot for EEA1, Rab5, LAMP1 and IL-1β. Arrows denotes a smaller product of EEA1 upon hypotonic stimulation. IL-1β released to the extracellular medium was also detected by ELISA (bottom). (B) Immunoblot analysis of EEA1 and caspase-1 on cell lysate and cell-free supernatant from LPS-primed THP-1 (wild-type, CASP1−/− or CASP4−/−) after hypotonic stimulation. Where indicated, Ac-YVAD was added to inhibit caspase-1. (C) In vitro processing assay for recombinant human EEA1 expressed in HEK293 cells and incubated with either recombinant caspase-1, or with concentrated supernatant or cell lysates from wild type (WT) or Casp1/11−/− LPS-primed BMDM activated with nigericin. Where indicated, Ac-YVAD was added to inhibit caspase-1. Dashed line separates two different blots. (D) In vitro processing assay for recombinant human EEA1 WT, D132A, D127A, or D127A/D132A mutants expressed in HEK293 cells and incubated with recombinant caspase-1. (E) Representative high resolution deconvolved images of LPS-primed BMDMs after incubation with hypotonicity and stained with fluorescent probe for active caspase-1 (FLICA, green) and the endosome marker EEA1 (red) and nuclei (DAPI, blue); bar, 5 µm. (F) Co-immunoprecipitation of EEA1 and the p10 subunit of active caspase-1 in cell supernatants of LPS-primed THP-1 macrophages after NLRP3 stimulation with hypotonicity. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete membrane fragments are showed in all cases. Blots and pictures are representative of three to four independent experiments. ELISA data are presented as average ± sem from n = 3 independent experiments.