Fig. 4: Calpain mutant resistant to KCTD7-mediated ubiquitination is hyperactive. | Cell Discovery

Fig. 4: Calpain mutant resistant to KCTD7-mediated ubiquitination is hyperactive.

From: Calpain activity is negatively regulated by a KCTD7–Cullin-3 complex via non-degradative ubiquitination

Fig. 4

a In vivo ubiquitination assay for calpain1 performed by expressing WT KCTD7 or pathogenic mutants in HEK293 cells for 36 h. b In vitro ubiquitination assay for calpain 1. c In vivo ubiquitination assay in HEK293 cells for calpain 1 K–R mutants. d In vivo ubiquitination assay for calpain 1 K–R mutants performed with or without KCTD7 in HEK293 cells. e, f Co-IP analysis of Myc-tagged CAPNS1 and Flag-tagged WT calpain 1 or calpain 1-K398R in calpain 1 knock-out HEK293 cells, n = 5. g Confocal microscopy of HeLa cells showing distribution of WT calpain 1 or calpain 1-K398R. Na+/K+-ATPase was used as plasma membrane marker. Scale bar, 10 μm. h–j WT calpain 1 or K398R mutant were expressed in calpain 1 knock out Hela cells and cleavage of α-spectrin cleavage and caspase 3 were evaluated by immunoblotting, n = 5. Data represent means ± SEM; ∗P < 0.05, ∗∗P < 0.01, ns not significant.

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