Fig. 3
CKIP-1 regulates the stability of Oct-1 via REGγ-dependent proteasome degradation. a Co-IP of Oct-1 and CKIP-1. HA-Oct-1 and Myc-CKIP-1 were transfected into HEK293T cells. IP immunoprecipitation, WCL whole cell lysate. b Co-IP of CKIP-1 and REGγ. Flag-REGγ and Myc-CKIP-1 were transfected into HEK293T cells. c Co-IP interaction assay for the endogenous interaction between CKIP-1 and REGγ in BMDMs. d Immunofluorescence assays show the co-localization of CKIP-1 and REGγ in BMDMs. Scale bar, 25 μm. e Co-IP of Oct-1 and REGγ. Myc-REGγ and HA-Oct-1 were transfected into HEK293T cells. f Co-IP interaction assay for the endogenous interaction between REGγ and Oct-1 in BMDMs. g HA-Oct-1, Myc-CKIP-1, and REGγ siRNA were cotransfected into HeLa cells and proteins were analyzed by western blot. h HEK293T cells were co-transfected with HA-Oct-1 with increasing amount of Myc-REGγ as indicated. i HEK293T cells were transfected with HA-Oct-1, Myc-REGγ, Myc-CKIP-1 or together as indicated. j Knockdown of REGγ levels in WT and Ckip-1−/− macrophages transfected with REGγ-shRNA #1, #2 by lentivirus. k Knockdown of REGγ in WT macrophages followed by CHX (10 μg per ml) treatment for the indicated times. l The transfected HEK293T cells were treated with MG132 (20 μM) as indicated for 8 h before harvest, and cell lysates were subjected to western blot. m HEK293T cells were transfected with HA-Oct-1 and WT REGγ or N151Y mutant, and cell lysates were subjected to western blot. n HEK293T cells were transfected with HA-Oct-1, Flag-CKIP-1, and Flag-REGγ N151Y mutant as indicated, and Co-IP assays were performed. o HeLa cells were transfected with siRNA against REGγ or Oct-1 as indicated and then cells were transfected with Renilla luciferase plasmids together with LOX-1/pGL3 luciferase plasmid. p Luciferase assay in HEK293T cells were transfected with the LOX-1/pGL3 luciferase plasmid and CKIP-1, Oct-1, REGγ plasmids as indicated. Data represent mean ± s.e.m. of n = 3 biologically independent experiments (k, o, p). P values were calculated by two-way ANOVA (k) and two-tailed Student’s t-test (o, p). *P < 0.05, **P < 0.01, ***P < 0.001. The precise P value and statistics source data are shown in Supplementary Data 2. Unprocessed original scans of blots are shown in Supplementary Fig. 6
