Fig. 6: LIN28 recruits MSI2 to regulate YAP1 activation.

A RNA immunoprecipitated (RIP) with anti-Flag antibody followed by qRT-PCR to analyze LIN28 binding of YAP1 and TAZ mRNAs, and other indicated target mRNAs in MCF-10A cells stably expressing Ctrl or Flag-LIN28A. The enrichment folds of mRNAs present in the LIN28 IP are relative to the control IP. The experiment was done in 3 biological replicates and data from a representative experiment are shown. B Consensus motifs within LIN28 clusters identified in ESCs and 293T cells, and the mapped GGAGA motifs in indicated genes are shown with green triangle. C WB analyses of total proteins (Input) or anti-Flag antibody immunoprecipitated (Flag-IP) proteins from CAL51 cells stably expressing vector control (Ctrl) or Flag-LIN28A/B using the indicated antibodies. D WB analyses of Input or Flag-IP proteins from CAL51 cells stably expressing Ctrl or Flag-MSI2 using the indicated antibodies. E, F Co-IP showing the interaction between endogenous LIN28A and MSI2 in CAL51 cells, or exogenous LIN28A and MSI2 in 293T cells. G, H Schematic of full-length LIN28A and MSI2 protein domains and their truncated constructs. I Co-IP analysis to determine the regions of LIN28A required for interacting with MSI2, and the regions of MSI2 required for interacting with LIN28A. J, K WB analyses of total proteins from CAL51 cells stably transfected with Ctrl, Flag-MSI2, or ShCtrl, ShMSI2-1, and ShMSI2-2 using the indicated antibodies. L WB analyses of puromycin-labeled nascent proteins in CAL51 cells stably expressing the indicated vectors. Puromycin (10 μg/ml) was added to the culture medium of CAL51 cells 30 min before harvest (with and without serum starvation), Actin was detected as an internal control.