Fig. 4
LATS1/2 directly interacts with STAT1 and phosphorylates STAT1. (A, B) Western blotting of the WCLs and co-IP samples of anti-FLAG antibody obtained from 293T cells transfected with the indicated plasmids. (C-E) Immunoprecipitation using anti-LATS1 (C), anti-LATS2 (D), or anti-STAT1 (E) antibodies in the WCLs prepared from KLE cells followed by western blotting with the indicated antibodies. (F) Recombinant expressed GST-STAT1 protein or GST bound to glutathione-Sepharose beads and incubated with recombinant expressed His-LATS1 or LATS2 proteins. Bound His-LATS1 or LATS2 proteins were detected by western blotting with anti-His antibody. (G, H) Recombinant GST-STAT11 − 650aa or GST-STAT1650 − 750aa proteins were subjected to in vitro phosphorylation by active human His-LATS2480 − 1088aa protein. The MS spectra correspond to two phosphorylated STAT1 peptides are shown. (I) Recombinant GST-STAT1 proteins were subjected to phosphorylation by active human His-LATS2480 − 1088aa protein, as detected using in vitro kinase assays. The reaction products were subjected to western blotting. (J) Recombinant GST-STAT1 proteins were subjected to phosphorylation by active human His-LATS1589 − 1130aa protein, as detected using in vitro kinase assays. The reaction products were subjected to western blotting. (K) Western blotting of the indicated proteins in the WCLs from parental, LATS1-KO, LATS2-KO or LATS1/2-KO KLE cells. (L) Western blotting of the indicated proteins in WCLs from KLE cells treated with TDI-011536 (3 µM) for 24 h. (M) Exogenous LATS1 (WT or kinase dead mutant) was reintroduced into LATS1/2-KO KLE cells to generate LATS1 Tet-On inducible cells. The cell lines were treated with DMSO or doxycycline (DOX) (10 ng/ml) for 24 h, and the WCLs were prepared for western blotting. (N) Exogenous LATS2 (WT or kinase dead mutant) was reintroduced into LATS1/2-KO KLE cells to generate LATS2 Tet-On inducible cells. The cell lines were treated with DMSO or DOX (10 ng/ml) for 24 h, and the WCLs were prepared for western blotting. (O, P) Representative IF images from 293T cells transfected with the indicated plasmids and stained with LATS1 (FLAG), LATS2 (FLAG), STAT1 (pEGFP) and DAPI (O). Scale bar, 20 μm. Quantification of the ratio of STAT1 in Nuc (nucleus)/Cyto (cytoplasm) is shown in (P). Data are shown as means ± SD (n = 10). (Q, R) Representative IF images from parental and LATS1/2-KO KLE cells treated with IFN-γ (30 ng/ml) for 6 h, stained with STAT1 and DAPI (Q). Scale bar, 20 μm. Quantification of the ratio of STAT1 in Nuc/Cyto is shown in (R). Data are shown as means ± SD (n = 20). P values are calculated using One-way ANOVA test in (P, R). *p < 0.05, ****p < 0.0001