Fig 2: SLP65 regulates PTEN localization through RHOA. (A) Left- Wild type (WT) bone marrow derived pre-B cells were treated with RHO-specific inhibitor C3-toxin (RHO I) for 16 hours or were left untreated for 16 hrs. Total cell lysates were prepared and immunoblotting was performed. Data are representative of 3 independent experiments. Right- quantification of the western blots. RhoA and PTEN blots are normalized with GAPDH while p-AKT is normalized with total AKT. Bars represent mean ± SD. Statistical test- one-way ANOVA with multiple comparison test. (B) SLP65 deficient pre-B cells were retrovirally reconstituted with ERT2-SLP65 and were induced with 4-OHT for 30 min with or without RHO (I) Cells treated with vehicle were used as a control. Cells were then fixed with 2% paraformaldehyde and labeled with DyLight 488 conjugated anti-PTEN antibody and DAPI staining for immunofluorescent microscopy. Images were captured by using a Zeiss LSM 780 laser scanning confocal microscope with GaAsP detectors and visualized with Imaris software. *p<0.05, **p<0.01, ns, not significant.

Fig 3: SLP65 interacts with RHOA and regulates its activation. (A) Left- PLA showing the association of SLP65 and RHOA in DG75EB/HA-RhoA cell line. Only secondary antibodies are used in the controls. Close proximity is shown as red dots. Right- quantification of number of signals per cell, error bars represent mean ± SD. Unpaired t-test, two-tailed. (B) Left- PLA showing the association of RHOA with phosphorylated SLP65 in DG75EB/HA-RhoA cell line upon BCR stimulation. Cells were stimulated with anti-IgM F(ab)2 antibody for the specified time period. Right- quantification of number of signals per cell, bars represent mean ± SD. Unpaired t-test, two-sided. (C) Detection of total cellular RHOA activity by G-LISA RHOA activation assay. Reconstituted bone marrow derived pre B cells from TKO mice were treated with ethanol (control) or 4-OHT to induce SLP65 for the indicated time point. Equal amounts of cell lysates from control and 4-OHT treated cells were used for the assay. Statistical analysis: one sample t test. (D) Western blot analysis for the detection of RHOA upon SLP65 activation in TKO cells. The cells were reconstituted with µ-heavy chain, ?5 and inducible SLP65-ERT2 construct and then induced with 2µM 4-OHT or Et for 30 min to activate SLP65. GAPDH is used as loading control. *p<0.05, ***p< 0,001, ****p<0.0001, ns, not significant.