Fig 1: 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