Fig. 5
From: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
USP8 binds and deubiquitinates trichoplein. a Bacterially purified GST or GST-USP8 were incubated with RPE1 cell lysates, and then affinity purified with glutathione-sepharose. The samples were analyzed by immunoblotting with anti-trichoplein and by coomassie staining. Asterisk indicates fragmented GST-USP8. b Endogenous trichoplein (indicated by arrow) was co-immunoprecipitated with anti-FLAG antibody form TetOn-RPE1 FLAG-USP8 WT cells that were treated with, but not without, doxycycline (Dox: 100 ng ml−1). c GST-USP8 was incubated with MBP-trichoplein in vitro, and then affinity purified with glutathione-sepharose. d Myc-trichoplein and HA-ubiquitin were co-transfected with or without FLAG-USP8 (WT, S718A, or C786S) in HEK293T cells. Six hours after treatment with 10 μM MG132, anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-HA and anti-Myc. Amounts of FLAG-USP8 proteins were analyzed in cell lysates. e Polyubiquitinated Myc-trichoplein ([HA-Ub]n-Myc-trichoplein) was immunoprecipitated from HEK293T cells transfected with Myc-trichoplein and HA-ubiquitin (input), as described in d, and then incubated with GST or GST-USP8 (WT or C786S) for 1 h at 37 °C
