Fig. 2
From: APC/CCDH1 synchronizes ribose-5-phosphate levels and DNA synthesis to cell cycle progression
APC/CCDH1 controls TKTL1 proteasomal degradation. a TKTL1 levels in HeLa cells were determined at different time points after protein synthesis was blocked by cycloheximide. b TKTL1 levels were determined in HeLa cells cultured with or without the proteasome inhibitor MG132. c TKTL1 and TKT ubiquitination. d The TKTL1 sequence matching the D-box consensus sequence and the TKT sequence corresponding to the TKTL1 D-box sequence are shown. e Co-immunoprecipitation of TKTL1-FLAG and CDH1-Myc co-expressed in HeLa cells. f Affinity purified TKTL1 from lysates of ccRCC tissue was probed for CDH1 to detect the in vivo interaction of TKTL1 and CDH1. g Endogenous TKTL1 levels were measured in HeLa cells and HeLa overexpressing CDH1 or CDC20 (n = 3 biologically independent samples). Quantitation of western blots is shown on the right. Error bars, mean ± SEM, Student’s t test. ***p < 0.001, ns not significant. h FLAG-TKTL1, Ub-HA, and CDH1-Myc were co-expressed in HeLa cells and then TKTL1 was purified by immunoprecipitation. Ubiquitination levels of TKTL1 were determined by anti-HA antibody. i TKTL1-FLAG, Ub-HA, and CDH1 siRNA were co-transfected in HeLa cells. Ubiquitination levels of immunoprecipitated TKTL1 were then detected. j Two siRNAs targeting different CDH1 regions were used. Representative western blot and mean values (n = 3 biologically independent samples) of quantitative western blotting results are shown. Mean values with SEM are reported, Student’s t test. **p < 0.01, ns not significant. k A commercial ubiquitination system was employed to detect ubiquitination of purified TKTL1 by APC/CCDH1. l Amounts of CDH1 co-immunoprecipitated with TKTL1 and TKTL1ΔD-box were compared when they were co-expressed in HeLa cells at comparable levels. m Ubiquitination levels of TKTL1 and TKTL1ΔD-box were detected after they were expressed alone or co-expressed with CDH1 in HeLa cells. n TKTL1 and TKTL1ΔD-box levels in HeLa cells were detected in the presence or absence of CDH1 co-expression in cells. o Endogenous TKTL1 levels were determined in TKTL1ΔD-box-knockin HeLa cells and TKTL1ΔD-box-knockin HeLa overexpressing CDH1 or CDC20. p Ubiquitination levels of affinity purified TKTL1-FLAG at different cell cycle phases were determined. Full-length blots are presented in Supplementary Fig. 10
