FIGURE 3.
Numb isoforms with shorter PTB facilitate NPC1L1 internalization in mammals. A, schematic of Numb isoforms in mammals. Lengths of their PTB and PRR domains are various. B, the mRNA expressions of Numb isoforms with different PTB domains analyzed by semi-quantitative RT-PCR in mouse tissues. Primers used were indicated in A. SPTB, shorter PTB; LPTB, longer PTB. C, the mRNA expressions of Numb isoforms with different PTB domains in human liver and intestine (two samples of each tissue). D, purified His6-tagged shorter and longer Numb-PTB proteins from E. coli. E, in vitro pulldown assay of Numb-PTB with GST-NPC1L1-C (WT or Y1306A). After incubation of GST fusion proteins with Numb-PTBs, the pellets and inputs were assessed by Western blots with the indicated antibodies. F, internalization of NPC1L1 in Numb RNAi cells transfected with plasmids encoding Myc-tagged Numb isoforms (marked by asterisk). The sequences of Numb isoforms in plasmids were siRNA-resistant. The cells were treated as described in the legend Fig. 2B and immunofluorescent staining was performed to label exogenous Numb. Scale bars, 10 μm. IB, immunoblot.