Fig 2: UbiScan for global identification of LIN41 ubiquitination substrates. (a) Proteomic strategy for global identification of LIN41-mediated ubiquitination targets under self-renewal conditions (LIF) and during neural differentiation with RA using a mouse ES cell line with Dox-inducible expression of LIN41-Flag. Each extract was processed by digestion with trypsin followed by affinity purification using an antibody specific for lysine residues carrying a diglycine linkage (K-GG). Enriched ubiquitin-modified peptides were analyzed by spectrometry. (b) iLin41 protein extracts prepared for the UbiScan were immunoblotted with α-LIN41 and α-OCT-4 antibodies to validate LIN41 induction by Dox and the conversion of pluripotent ES cells to differentiated cells after 3 days of RA treatment, respectively. (c) Venn diagram compares the sets of proteins showing a ≥2.5-fold increase in ubiquitination after LIN41 induction between the pluripotent and differentiated cell states. (d and e) Functional annotation analysis depicting significantly enriched Panther pathways among proteins with ≥2.5-fold more ubiquitination upon LIN41 induction in pluripotent (d) and differentiated (e) cells. Similar results were obtained for enrichment of p53-related KEGG or REACTOME pathways using Gene Set Enrichment Analysis (http://software.broadinstitute.org/gsea/msigdb/index.jsp, data not shown). (f) Diagram summarizing selected p53 pathways with LIN41 ubiquitination candidates from UbiScan depicted in blue and their functional relationships (arrow=direct activation, dotted arrow=indirect activation, inhibition line=direct negative regulation). (g) Fold change ratio of Dox+ versus Dox− in differentiated cells for the indicated diglycine-modified sites within p53 and schematic of p53 domain structure. DBD, DNA Binding Domain; NLS, Nuclear Localization Sequence; TAD, Transactivation Domain; TET, Tetramerization domain. See also Supplementary Figure 1 and Supplementary files 1–4

Fig 3: Comparison of IgG levels against CA15-3 (A), CEA (B), CA19-9 (C), c-Myc (D), p53 (E), Hsp27 (F) and Hsp70 (G) were measured in normal, CIN I, CIN II, CIN III and cancer groups. The procedure for detecting IgGs against relevant TAAs by ELISA is described in Materials and Methods. Central lines are mean values, and error bars show ranges of SD values. Normal, n=28; CIN I, n=28; CIN II, n=30; CIN III, n=31; Cancer, n=31. Numbers in parenthesis are mean values.P-values were calculated from the Mann-Whitney-U test. The Bonferroni correction was performed, and p<0.05 was considered statistically significant (*p<0.05; **p<0.01; ***p<0.001).

Fig 4: LIN41 co-localizes and physically interacts with p53. (a) iLin41 cells were immunostained for endogenous p53, LIN41 and nuclear marker Draq5. (b) Flag-tagged LIN41 co-precipitates endogenous p53 in HEK293 cells. Co-precipitation of endogenous proteins after IP with either control IgG or α-Flag agarose was demonstrated by western blotting. (c) Flag-tagged p53 co-purifies endogenous LIN41. P19 cells were transfected with Flag-p53 or positive control Flag-Mov10. After α-Flag-IP, LIN41 co-purified with similar efficiency using either MOV10 or p53 as bait. (d) p53 from mouse ES cells was affinity purified using a mouse-specific p53 Trap. A human-specific p53 Trap served as negative control. Endogenous interaction was observed between p53 and LIN41, but not with GAPDH. (e) Schematic representation of LIN41 domains and the deletion constructs used to map the p53 interaction. (f) p53 interacts with the NHL domain of LIN41. LIN41-GFP fusion proteins containing the full-length protein or deletion constructs without the NHL, the RING motif or with only the NHL domain were co-expressed with Flag-tagged p53 in HeLa cells. GFP was included as a negative control. Input and bound proteins after Flag-IP were analyzed by western blotting with the indicated antibodies. (g) p53 directly interacts with LIN41. GST pull-down assays of in vitro synthesized LIN41 using GST only, p53-GST fusion protein or MDM2-GST. The presence of LIN41 in bound and unbound fractions was analyzed by immunoblotting. See also Supplementary Figure 2

Fig 5: Loss of LIN41 elevates p53 steady-state levels and reduces p53 ubiquitination. (a) Comparison of p53 expression in undifferentiated wild type, heterozygote and homozygote Lin41-mutant ES cells shows a gene dose-dependent increase of p53 protein levels in Lin41-deficient cells. (b) Lin41 deficiency in mouse ES cells does not affect p53 transcript levels analyzed by qRT-PCR. Expression levels were normalized to Oaz1 mRNA. Data are presented as means±S.D. values from three independent clonal ES cell lines per genotype. (c) Constitutive levels of the p53 effector Noxa are increased in Lin41-deficient ES cells. Expression levels of Puma, Noxa and Bim were measured by qRT-PCR and normalized to Oaz1. Data are presented as means±S.D. values from three independent clonal ES cell lines per genotype. **P<0.01. (d) Cre-induced disruption of LIN41 in Lin41fl/fl; Rosa26-CreERT2 mES cells upregulates p53 steady-state levels. Conditional Lin41 knockout cell lines were cultured under proliferative and differentiation conditions. For the induction of Cre-mediated recombination, cells were treated with 500 nM 4-hydroxytamoxifen. p53, OCT-4 and LIN41 protein expression were examined in untreated cells (lanes 1 and 5), in two independent cell lines with stable Lin41 deletion (lanes 2 and 6 plus 3 and 7) or acute loss of Lin41 (lanes 4 and 8). (e) Quantification of p53 levels in two independent lines with stable Cre-induced Lin41 deletion relative to untreated cells. Relative band intensity was quantified from immunoblots from three independent experiments. Data are presented as means±S.D. values. *P<0.05, ***P<0.001. (f and g) Loss of LIN41 reduces p53 ubiquitination in vivo. After 4 h MG132 treatment, total ubiquitinated proteins from 2-day RA-treated Lin41fl/fl; Rosa26-CreERT2 cells were enriched using α-Ub-FK2-agarose beads or TUBEs, as indicated. Ubiquitination levels of endogenous p53, LIN41 and pull-down efficiency were detected by immunoblotting