Fig 1: Disorder Is the Key Feature of Degraded Proteins(A) Diagrams of human MTMR1 and D2HGDH (above) and corresponding predicted disorder plots (below) (Hanson et al., 2017).(B) HEK293T cells were transiently transfected with plasmids encoding either the full-length or N-terminally truncated, disorder free (?N-term), indicated constructs (2 µg). After 16–20 h, cells were harvested, lysed, and incubated with varying concentrations of trypsin (20, 2, and 0.2 ng/µL) or PROK (4, 0.8, 0.16, and 0.032 ng/µL) before immunoblotting.(C) HEK293T cells were transiently transfected with plasmids encoding the full-length or N-terminally truncated indicated constructs (0.1 µg). After 16–20 h, cells were treated with VbP (10 µM) for 18 h before protein levels were evaluated by immunoblotting.(D and E) HEK293T cells stably expressing human CASP1 and GSDMD were transiently transfected with plasmids encoding the indicated constructs (0.02 µg). After 16–20 h, samples were treated with VbP (10 µM) for 6 h before cell viability was evaluated by LDH release (D) and lysates were evaluated by immunoblotting (E).(F and G) THP-1 CARD8-/- monocytes stably expressing the indicated constructs were treated with compound 8j (20 µM) or VbP (10 µM). After 24 h, cell viability was evaluated by LDH release (F) and lysates were evaluated by immunoblotting (G).(H) Schematic of the proposed CARD8 activation mechanism, which involves the degradation of its disordered N-terminus.Related to Figure S4.
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