Fig. 5: RFC4 binds to stabilize NICD1 by abrogating CDK8/FBXW7-induced degradation.
From: An RFC4/Notch1 signaling feedback loop promotes NSCLC metastasis and stemness
a MS peptide sequencing immunoprecipitated components, using anti-FLAG affinity purification, from lysates of A549 cells transfected with FLAG-tagged RFC4. b Immunoprecipitation assay revealing the interaction between RFC4 with NICD1. c SPR analysis measuring the affinity and kinetics of the interaction between RFC4 with NICD1 and CDK8 with NICD1. NICD1 was immobilized on a CM5 chip. d Lysates of 293FT cells transfected with NICD1-HA were immunoprecipitated by anti-HA affinity gel and incubated with purified RFC4 or CDK8 proteins, followed by addition of the indicated doses of purified CDK8, RFC4 proteins. The resultant incubates were analyzed by WB with the indicated antibodies. e The interaction between NICD1 and CDK8 or RFC4 in the presence or silencing of RFC4 or CDK8 was evaluated by immunoprecipitation of NICD1. f, g WB analysis of the effect of overexpressing or silencing RFC4 or together with CDK8 silencing on serine and threonine phosphorylation levels of immunoprecipitation pull-downed NICD1. h The interaction between NICD1 and CDK8 or FBXW7 in the presence or absence of RFC4-FLAG was evaluated by immunoprecipitation of Notch1. i The interaction between NICD1 and FBXW7 in the presence or absence of RFC4-FLAG was evaluated by immunoprecipitation of NICD1-HA in A549 WT and FBXW7 knockout cells. j, k The levels of K48-linked polyubiquitination of NICD1 were evaluated by immunoprecipitation of FLAG-tagged NICD1 in A549, H1975, LC1, and LLC cells with the indicated treatments. Source data are provided as a Source Data file, except Fig. 5c.
