Fig 1: SFK overexpression suffices to drive ligation-independent positive and negative signaling pathways. (A) Lck-expressing cells have more robust signaling responses on a whole cell population basis. Resting Lck- or Lyn-expressing cells were stained for pCD79a, pSyk (Y525/526) and pAKT (Ser473) and analyzed by FACS. Graphs display the frequency of cells double-positive for GFP and pCD79a, pSyk and pAKT respectively (Gating as in Figure 1A , % of cells in the upper right quadrants) (n≥4, Unpaired Student t test; mean +/- standard deviation [SD]; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant). (B) Lyn is more efficient than Lck in igniting phosphporylation of BCR signaling mediators on an equal protein expression level. Same as in (A) but for equivalent levels of SFK expression ( Figure S3E for description of equal GFP gating strategy). (C) Enhanced CD22 tyrosine phosphorylation in response to SFK overexpression. CD22 was immunoprecipitated from the Lck- and Lyn-BJAB cell lines and their -Dox counterparts in the absence or presence of stimulation with 10 µg/mL a-IgM for 10min. IPs were analyzed by western blotting with a-pY 4G10 (upper panel) and a-CD22 (lower panel) antibodies. HEK293T cells (devoid of CD22 expression) were used as a negative control for the IPs. (D) SFK overexpression ignites the CD22/SHP-1 inhibitory pathway and this depends on their intact kinase activity. Left-hand panels. CD22 IPs as in C, but with additional samples treated for 10 min with 100 µM of the pan-SFK inhibitor PP2, as indicated. IPs were analyzed by western blotting with a-pSHP-1 Y564 (upper panel), total a-SHP-1 (middle panel) and a-CD22 (lower panel) antibodies. Right-hand panels. Total cell lysates from the same experiment, analyzed by western blotting with a-pSHP-1 Y564 (upper panel) and a-pY416 (middle panel) to verify the PP2-induced reduction in SFK activity. Actin blot (lower panel) attests equal sample loading. Shown are representative blots of two independent experiments.