Figure 4
From: Fat3 regulates neural progenitor cells by promoting Yap activity during spinal cord development
Fat3 knockdown destabilizes and attenuates Yap transcriptional activity. (A) P19 cells were transfected with sh-control and sh-Fat3 (target sites #1 and #2), followed by RT-PCR to test knockdown efficiency. (B) P19 cells were transiently transfected with sh-Fat3 and Fat3ΔN together with Flag-Yap, HA-Lats2 and HA-Mst2. After 48 h, cell lysates were analyzed by western blotting. (C) Schematic representation of the mouse Fat3 full length and N-terminus deleted construct of Fat3 (ΔN). (D) The phosphorylation level of Yap by Lats2/Mst2 was attenuated by overexpression of Fat3ΔN. anti-S127P Yap antibody detects the phosphorylated Serine 127 residue of Yap. anti-T1041P, T1079P Lats1/2 antibody detects the phosphorylated Lats2 on Thr1041 and Thr1079. Quantification of each signal measured by ImageJ and then normalized by tubulin is shown below each band. (E) Luciferase reporter assay with UAS:LUC and Gal4-Tead showed the transcriptional activity augmented by Yap, that was abrogated by Fat3 knockdown. Values are means ± sd. At least 3 replicate experiments were repeated in P19 cells. (F,G) Fat3 interacts with Lats2 but not with Yap. Co-immunoprecipitation assay with HEK293T cells transiently transfected with the expression vectors for HA-tagged Lats2 and Flag-tagged Fat3ΔN (F), and HA-tagged Fat3ΔN and Flag-tagged Yap (G), respectively. (H) CoIP assay with E11.5 mouse spinal cord extract showed interaction between Fat3 and Lats1. To detect same samples with different antibodies, membranes were cut prior to hybridization or reprobed. Uncropped original western blot images are included in the “Supplementary Data”, with cropped areas highlighted with colored boxes.
