Fig 2: Effects of BBR and M2 on Rasa3 membrane translocation upon ADP stimulation in vitro. The WPs were pretreated with the studied compounds or the vehicle control (0.1% DMSO) for 10 min, and stimulated with 10 µM of ADP for 10 min. After treatment, the cytosolic and membrane proteins were extracted for Western blot to detect Rasa3, Rap1, ERK (cytosolic control protein), and FcR? (membrane control protein). (A-B) The WPs were pretreated with BBR or the vehicle control as indicated. (C-D) The WPs were pretreated with M2 or the vehicle control as indicated. (E-F) The WPs were pretreated with BBR (0.5 µM), M2 (0.5 µM), Wtm (100 nM), or the vehicle control as indicated. Representative blots are presented (A, C, E). After quantification, the expression levels of cytosolic and membrane Rasa3 were normalized to those of ERK and FcR?, respectively, and plotted as indicated (B, D, F). Data are mean ± SD of three or four independent experiments. Statistically significant differences compared with ADP group are indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and statistically significant differences between the BBR and M2 groups are indicated by $ p < 0.05.

Fig 3: Talin1 is involved in cell polarity downstream of Rap1(A) Changes of cell elongation of naive WT and Tln1−/− T cells after stimulation. T cells suspended in RPMI1640 containing 1% BSA were stimulated with CCL21 (100 nM) and examined for cell elongation by imaging cytometry. The data shows the average percentage ±SD (n = 3, a representative of two experiments).(B) Changes in the cell polarization of naive WT and Tln1−/− T cells as in (A).(C) Chemotactic assays of WT, Tln1−/− (left panel) and Rap1a−/−Rap1b−/− (right panel) T cells in response to 30 nM CCL21 (n = 3).(D) The amount of RhoA-GTP of Tln1−/− T cells at 2 min after stimulation with CCL21 (100 nM) measured by flow cytometry. Bars indicate the average MFI of RhoA-GTP ±SD (n = 3, a representative of two experiments).(E) The amount of pMLC of Tln1−/− T cells at 2 min after stimulation with CCL21 (100 nM). The relative amount of pMLC in mutant T cells was calculated as MLC levels by MFI normalized to the average of MFI of unstimulated WT T cells. (n = 3, a representative of two independent experiments).(F) The clustering of pMLC in WT and Tln1−/− T cells at 2 min after stimulation with CCL21 (100 nM) was measured by imaging cytometry. Bars indicate the average ±SD (n = 3, a representative of two independent experiments).(G) A representation of confocal images of F-actin, talin1, and pMLC in HT-talin1 knock-in (Tln1HT/WT) T cells stained with HaloTag SaraFluor 650T Ligand. WT T cell (Tln1w/w) was used for negative control of HT-specific staining. Scale bar, 5 μm.(H) Relative levels of pMLC in WT, Rasa3−/−Sipa1−/−, Rasa3−/−Sipa1−/−Tln1−/−, and Tln1−/−T cells were measured by flow cytometry. Bars indicate average ±SD (n = 3, a representative of two independent experiments).(I) The clustering of pMLC (average ±SD) as in (H).(J) The cell elongation (average ±SD) as in (H). Statistical significance for the above data was determined by two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.