Fig. 3

Acetylation of Lin28B by PCAF and its effect on the level of let-7. a In vitro acetyltransferase assay. GST-PCAF was incubated with GST-Lin28B in the presence of CoA, and blotting with antibodies against pan-acetyl lysine (AcK) and GST. * indicates autoacetylated PCAF-HAT and its derivatives. b Acetylation of Lin28B by PCAF is HAT domain-dependent. WCEs of HEK293T cells co-transfected with FLAG-Lin28B and either Myc-PCAF or Myc-PCAF-△HAT were IP-ed with anti-FLAG, then blotting with anti-AcK . and anti-FLAG. c Effect of PCAF on let-7. H1299 cells were transfected with FLAG-PCAF, FLAG-PCAF-△HAT or the vector. The expression levels of FLAG-PCAF and FLAG-PCAF-△HAT are shown at lower panel. RT-qPCR showing the levels of let-7a-1, let-7g and mir-16-1. Each bar represents the mean value ± S.D. from at least three independent experiments (#, P > 0.05; *p ≤ 0.05; **p ≤ 0.01, upper panel). d Interaction of PCAF with Lin28B is not dependent on the HAT domain. WCEs of FLAG-PCAF or PCAF-△HAT-transfected cells were IP-ed with anti-FLAG, then blotted with anti-FLAG and anti-Lin28B. IP with IgG served as a control. e Nuclear (NE) and cytoplasmic (CE) distribution of Lin28B. The extracts were blotted with anti-Lin28B. Lamin B: nuclear marker, GAPDH: cytoplasmic marker. f Localization of Lin28B and PCAF in H1299 cells. Immunofluorescence images showing ectopic Lin28B and PCAF in H1299 cells. g Effects of PCAF on the distribution of Lin28B. HeLa cells were co-transfected with FLAG-Lin28B and Myc-PCAF or vector (vec). CEs and NEs were immunoblotted with anti-FLAG. h The effects of PCAF on the distribution of Lin28B in H1299 cells. Cells were transfected with Myc-PCAF (wild-type, W; Cys574 mutant of PCAF, M) and FLAG-Lin28B (W, ΔCSD). CEs and NEs were immunoblotted with anti-FLAG and anti-Myc. i A schematic representation of the effect of PCAF-induced Lin28B acetylation on the synthesis of let-7