IndraLab

Statements

USP15 binds FF. 14 / 14
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"In contrast, inactive FF-USP15 CS redistributed primarily to intracellular punctate structures that were positive for HiBiT-CTLA4-FH ( xref )."
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"Consistent with this hypothesis, inclusion of the lysosomal (v-ATPase) inhibitor concanamycin A resulted in a marked rescue in the FF-USP15 CS protein levels and signal intensity ( xref )."
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"Lastly, we incubated cell lysates derived from FF-USP15 expressing cells with a reactive ubiquitin probe (Ubiquitin-Propargylamide, Ub-PA), which binds to WT but not CS mutant FF-USP15, producing a characteristic upshift in SDS–PAGE ( xref ) ( xref )."
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"Using our optimised protocol, we observed a 1.5 to 2-fold increase in HA-FRB*-c-Myc levels only upon Dox induction in FF-USP15 WT but not in CS expressing cells and only if treated for 24 h with the heterodimeriser A/C ( xref )."
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"We then transiently transfected this construct, CTLA4-FRB*-HA (CTLA4-FH), into FF-USP15 HEK293 cells (-Dox) and then inhibited translation with cycloheximide (CHX) to estimate protein half-life."
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"Induction of FF-USP15 WT, but not the CS mutant, imparts >2-fold increase in CTLA4-FH levels that is contingent on the A/C heterodimeriser ( xref )."
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"Reciprocal pulldown experiments of CTLA4-FH and FF-USP15 under these conditions demonstrate that ternary complex formation is comparable in WT and CS mutant cells ( xref ; note increased levels of CTLA4-FH in WT [+Dox, +A/C] input samples)."
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"For RapTag experiments scheduled according to the protocol shown in xref , overexpression of FF-USP15 WT or CS reach ∼40 to 50-fold the levels of endogenous USP15 at the point of lysis ( xref )."
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"Immunofluorescence microscopy confirmed that (i) most of the cells in the pool uniformly expressed the transgene after 24 h of induction and (ii) that FF-USP15 WT and CS mutant localise to both cytosolic and nucleoplasmic compartments as previously reported for endogenous USP15 ( xref ) ( xref )."
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"In agreement with our Western blot results ( xref ), we noticed an overall decrease in signal intensity for FF-USP15 CS in cells treated with A/C. We reasoned that this may reflect bystander degradation because of complex formation with CTLA4 and subsequent rapid shuttling of ubiquitylated CTLA4-USP15 CS heterodimers to the lysosome."
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"We first verified that this new construct can still be stabilised upon doxycycline induction of FF-USP15 WT, but not CS, in the presence of A/C ( xref )."
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"We, therefore, decided to establish a second set of RapTag cell lines, stably expressing HiBiT-CTLA4-FH alongside the Dox-inducible FF-USP15 ( xref )."
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"Applying a shortened Dox-induction schedule of 6 h followed by 18 h of A/C treatment, resulted in a ∼2-fold stabilisation (compared with −A/C) of HiBiT-CTLA4-FH in the FF-USP15 WT expressing cells ( xref )."
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"A/C treatment also caused a dramatic relocalisation of cytosolic WT FF-USP15 to the plasma membrane where it colocalised with HiBiT-CTLA4-FH ( xref )."
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