Figure 1.

Sharpin inhibits caspase activation and targets caspase-1 for linear ubiquitination. A, pLKO.1, shSharpin, and shHOIP HaCaT cells were stimulated with 50 mJ/cm² UVB and lysed at the indicated time points post-irradiation and subjected to immunoblot analysis. Data are representative of three independent experiments. B, caspase-1 activity was measured in the supernatant of pLKO.1, shSharpin, and shHOIP HaCaT cells following UVB irradiation using Caspase-Glo 1 assay. Data shown are representative of two independent experiments and are displayed as mean ± S.E. (error bars). ****, p < 0.0001. C, HaCaT cells were stimulated with 50 mJ/cm² UVB and harvested at the indicated time points post-irradiation. Lysates were subject to anti-caspase-1 immunoprecipitation, followed by immunoblot analysis. Data are representative of three independent experiments. D, HEK293T cells were transfected with equal amounts of the indicated plasmids. 24 h post-transfection, lysates were harvested and subjected to FLAG M2 immunoprecipitation and immunoblot analysis. E–H, as in D, but cells were lysed in 1% SDS denaturing lysis buffer, followed by dilution to 0.1% SDS for immunoprecipitation with either FLAG M2 affinity gel (E, G, and H) or anti-caspase-1 (F). Data are representative of two independent experiments, except H, which was performed once.