Figure 1.

SLP65 interacts specifically with GEF-H1 and triggers its activation. (A) Left panel- Immunoprecipitation followed by western blot analysis for the interaction of endogenous SLP65 with GEF-H1 in DG75EB/HA-RhoA cells upon BCR stimulation. Cells were stimulated with anti-IgM antibody for the indicated time period and IP was carried out with anti-SLP65 antibody. PLCγ2, a known interactor of SLP65 is used as a positive control. Result representative of 3 independent experiments. Right Panel- Quantification of immunoprecipitated GEF-H1 with respect to SLP65. Statistical analysis- One-way ANOVA. (B) Left- Proximity ligation assay (PLA) to detect the association of SLP65 and GEF-H1 in unstimulated 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. (C) PLA to detect the association of SLP65 and GEF-H1 in murine B cells isolated from bone marrow (BM) and spleen (SP) and human peripheral blood (PBL) B cells. B cells are isolated by CD19 magnetic sorting. Close proximity in a range of 10-40 nm is shown as red dots. Right- quantification of number of signals per cell, error bars represent mean ± SD. (D) Left- western blot analysis for the detection of phosphorylated GEF-H1 upon SLP65 activation in bone marrow derived early pre-B cells from Rag2^-/- , λ5^-/- , Slp65^-/- triple knock out (TKO) mice. The cells were reconstituted with µ-heavy chain, λ5 and inducible SLP65-ER^T2 construct and then induced with 2µM 4-hydroxy tamoxifen (4-OHT) or ethanol (Et) for 30 min to activate SLP65. GAPDH is used as loading control. Right- quantification of pGEF-H1 band intensity with respect to GAPDH. Result representative of 4 independent experiments. Statistical analysis- unpaired t test, two tailed. *p<0.05, ****p<0.0001, ns, not significant.