Fig 1: TTC36 reduces the binding of STK33 to HPD and subsequent STK33-mediated HPD T382 phosphorylation. Immunoblotting analyses were performed with the indicated antibodies a–i. a Immunoprecipitation analyses of LO2 cell lysates were performed with an anti-HPD antibody. b, c Ttc36 shRNA b or Flag-TTC36 c were stably expressed in LO2 cells. Immunoprecipitation analyses with an anti-HPD antibody were performed. d Phos-tag analysis was performed with bacterially purified His-STK33 and His-HPD or His-HPD T382A mutant in the presence or absence of ATP. e Ttc36 shRNA#1 (left panel) or Flag-TTC36 (right panel) were stably expressed in LO2 cells. The quantification numbers represent relative intensity of presented bands. f LO2 cells expressing Flag-HPD or Flag-HPD T382A mutant were treated with the indicated dosages of ML281 for 48 h. The quantification numbers represent relative intensity of presented bands. g LO2 cells expressing Stk33 shRNA#1 or #2 (left panel) with or without reconstituted expression of RNA-resistant(r) STK33 (right panel) were examined by immunoblotting analyses with the indicated antibodies. The quantification numbers represent relative intensity of presented bands. h, i HEK293T cells were transfected with the indicated plasmids. MG-132 (10 μM) was added 12 h before they were harvested with a guanidine–HCl-containing buffer. A Ni-NTA pull-down assay was performed
Fig 2: TTC36 deficiency in mice results in impaired learning and memory. a Mean escape latency per training block for the aged wild-type and Ttc36−/− mice (8–12 months, WT group: n = 13, Ttc36−/− group: n = 11), Mean ± s.e.m. b Mean escape latency for the aged wild-type and Ttc36−/− mice (8–12 months, WT group: n = 13, Ttc36−/− group: n = 10) during retention probe trial, Mean ± s.e.m. c Nest construction was quantified in aged wild-type and Ttc36−/−mice (8–12 months, WT group: n = 14, Ttc36−/− group: n = 7) mice. Data from panels a and c are analyzed by two-way ANOVA with Sidak’s post-hoc test. **P < 0.01. d Schematic for HPD degradation regulated by the TTC36-STK33-PELI1 signaling axis in tyrosinemia, inducing neurological damage and disorders
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