Fig. 6: Lamtor1-mediated motility is driven by Myosin II activity.

a MLC phosphorylation in the presence or absence of Lamtor1. WT-THP1, Lamtor1-KO-THP1, and Lamtor1-KO-Full-THP1 cells were lysed, and MLC phosphorylation was detected by western blotting with anti-p-MLC antibody (left). The concentration of p-MLC of WT-THP1 (white bar), Lamtor1-KO-THP1 (black bar), and Lamtor1-KO-Full-THP1 (mesh bar) in SDS-PAGE gel bands was determined using ImageJ, and statistical analysis was performed (right). b, c Myosin II-dependent Lamtor1-mediated motility. Chemotaxis of WT-THP1 (white bar), Lamtor1-KO-THP1 (black bar), Lamtor1-KO-Full-THP1 (mesh bar), blebbistatin-treated WT-THP1 (diagonal bar), and blebbistatin-treated Lamtor1-KO-Full-THP1 (dark mesh bar) in response to 25 ng/ml CCL2 was determined by Transwell assay (pore size, 5 μm) (b). Chemotaxis of WT-THP1 (white bar), Lamtor1-KO-THP1 (black bar), and Lamtor1-KO-THP1 expressing MLC-DD-HA, a constitutively active form of MLC (mesh bar) in response to CCL2 were evaluated by a Transwell assay (pore size, 5 μm) (left). Expression of MLC-DD-HA in Lamtor1-KO-THP1 was evaluated by western blotting using an anti-HA antibody (right) (c). d Active form of RhoA in WT and Lamtor1^−/− BMDCs. Cells were lysed, and GTP-bound RhoA was pulled down with GST-Rhotekin-RBD and detected by western blotting with an anti-mouse RhoA antibody. e MYPT1 phosphorylation in WT and Lamtor1^−/− DCs. WT and Lamtor1^−/− BMDCs were lysed and phosphorylated MYPT1 was evaluated by western blotting with an anti-p-MYPT1 antibody. Statistical analysis was performed by two-sided ANOVA with Tukey’s post hoc test (a–c) [means ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.001]. Data are representative of three experiments.