S unclear. To address one possible mechanism of RasGRP1 activation in response to CXCR4 signaling we carried out SDF1a stimulations of DN3 thymocytes in the presence of PI3K inhibitor, LY294002. RasGRP1 contains a C-terminal PT domain that has been shown to bind phosphoinositides and recruit RasGRP1 to the plasma membrane [32,33]. However, we found that inhibition of PI3K activity had no effect on ERK activation in response to SDF1a stimulation, suggesting PI3K mediated phoshoinositide generation is not required for RasGRP1 mediated ERK activation downstream ofCXCR4. Both RasGRP1 and RasGRP3 contain DAG binding C1 domains that have been classically MedChemExpress POR-8 thought to recruit RasGRPs to Ras containing membranes [18]. Therefore, it appears likely that the mechanism of RasGRP1 activation downstream of CXCR4 is PLCc dependent and involves RasGRP1 binding membrane DAG through its C1 domain. Interestingly, ERK activation downstream of CXCR4 is also defective in RAG22/2 DN3, unable to express a pre-TCR [38]. Since CXCR4 mediated ERK activation is pre-TCR dependent, RasGRP1 may activate ERK downstream of the pre-TCR. It is known that LAT is part of the pre-TCR signaling complex and that LAT can recruit and activate PLCc [39]. Given the potential for PLCc mediated DAG production downstream of the pre-TCR, we predict that RasGRP1 activation during b-selection is PLCc dependent. However, more experiments are needed to address the precise mechanism of RasGRP1/3 activation in thymocytes. The TCR signaling strength model of early T cell development suggests that the strength of the TCR signal transduced in DN3E thymocytes is the deciding determinant of ab vs cd lineage choice [40]. It is thought that strong tert-Butylhydroquinone signals transduced through the cdTCR lead to strong ERK activation and increased expression of EGRs and Id3, driving cd T cell selection [41]. In contrast, weak signals transduced through the pre-TCR, resulting in weak ERK activation, in conjunction with Notch and CXCR4 signals promote b-selection [4]. Interestingly, targeted Sos1 KO thymi show similar frequencies, but reduced numbers, of mature cd T cells compared to wild type thymi [26]. We found that RasGRP1/ 3 deficiency had no significant effect on frequencies or numbers of mature cd T cells. A recent report from Chen et al. confirmed thatRasGRP1 Is Required for b-SelectionFigure 7. DN3 require RasGRP1 for ERK phosphorylation in response to CXCR4 activation. A, B. Bulk thymocytes were pre-treated with (right panels) or without (left panels) 20 mM of the PI3K inhibitor LY294002 and were stimulated with 10 nM SDF1a for 0, 1, 2, 3 or 5 minutes. A. Induction of p-ERK (pT202/pY204) was measured in DN3 (CD42CD82Thy1.2+CD3lo Thy1.2+CD442CD25+) from B6 and DKO thymi. B. Induction of pAKT (pS473) was measured in DN3 (CD42CD82Thy1.2+CD3lo Thy1.2+CD442CD25+) from B6 and DKO thymi. C. Bulk thymocytes were stimulated with 10 nM SDF1a for 0, 1, 2, 3 or 5 minutes and the induction of p-ERK (pT202/pY204) was measured in DN3 (CD42CD82Thy1.2+CD3loRasGRP1 Is Required for b-SelectionThy1.2+CD442CD25+) from B6, 1KO, 3KO and DKO thymi. Data are representative of between 3 and 7 independent experiments. * represents a significant statistical comparison between B6 and DKO. # represents a significant statistical comparison between B6 and 1KO. */# p,0.05. doi:10.1371/journal.pone.0053300.gRasGRP1 deficient thymi show uninterrupted cd T cell development, further suggesting that RasGRP1 is dispensable for thymic cd T cell devel.S unclear. To address one possible mechanism of RasGRP1 activation in response to CXCR4 signaling we carried out SDF1a stimulations of DN3 thymocytes in the presence of PI3K inhibitor, LY294002. RasGRP1 contains a C-terminal PT domain that has been shown to bind phosphoinositides and recruit RasGRP1 to the plasma membrane [32,33]. However, we found that inhibition of PI3K activity had no effect on ERK activation in response to SDF1a stimulation, suggesting PI3K mediated phoshoinositide generation is not required for RasGRP1 mediated ERK activation downstream ofCXCR4. Both RasGRP1 and RasGRP3 contain DAG binding C1 domains that have been classically thought to recruit RasGRPs to Ras containing membranes [18]. Therefore, it appears likely that the mechanism of RasGRP1 activation downstream of CXCR4 is PLCc dependent and involves RasGRP1 binding membrane DAG through its C1 domain. Interestingly, ERK activation downstream of CXCR4 is also defective in RAG22/2 DN3, unable to express a pre-TCR [38]. Since CXCR4 mediated ERK activation is pre-TCR dependent, RasGRP1 may activate ERK downstream of the pre-TCR. It is known that LAT is part of the pre-TCR signaling complex and that LAT can recruit and activate PLCc [39]. Given the potential for PLCc mediated DAG production downstream of the pre-TCR, we predict that RasGRP1 activation during b-selection is PLCc dependent. However, more experiments are needed to address the precise mechanism of RasGRP1/3 activation in thymocytes. The TCR signaling strength model of early T cell development suggests that the strength of the TCR signal transduced in DN3E thymocytes is the deciding determinant of ab vs cd lineage choice [40]. It is thought that strong signals transduced through the cdTCR lead to strong ERK activation and increased expression of EGRs and Id3, driving cd T cell selection [41]. In contrast, weak signals transduced through the pre-TCR, resulting in weak ERK activation, in conjunction with Notch and CXCR4 signals promote b-selection [4]. Interestingly, targeted Sos1 KO thymi show similar frequencies, but reduced numbers, of mature cd T cells compared to wild type thymi [26]. We found that RasGRP1/ 3 deficiency had no significant effect on frequencies or numbers of mature cd T cells. A recent report from Chen et al. confirmed thatRasGRP1 Is Required for b-SelectionFigure 7. DN3 require RasGRP1 for ERK phosphorylation in response to CXCR4 activation. A, B. Bulk thymocytes were pre-treated with (right panels) or without (left panels) 20 mM of the PI3K inhibitor LY294002 and were stimulated with 10 nM SDF1a for 0, 1, 2, 3 or 5 minutes. A. Induction of p-ERK (pT202/pY204) was measured in DN3 (CD42CD82Thy1.2+CD3lo Thy1.2+CD442CD25+) from B6 and DKO thymi. B. Induction of pAKT (pS473) was measured in DN3 (CD42CD82Thy1.2+CD3lo Thy1.2+CD442CD25+) from B6 and DKO thymi. C. Bulk thymocytes were stimulated with 10 nM SDF1a for 0, 1, 2, 3 or 5 minutes and the induction of p-ERK (pT202/pY204) was measured in DN3 (CD42CD82Thy1.2+CD3loRasGRP1 Is Required for b-SelectionThy1.2+CD442CD25+) from B6, 1KO, 3KO and DKO thymi. Data are representative of between 3 and 7 independent experiments. * represents a significant statistical comparison between B6 and DKO. # represents a significant statistical comparison between B6 and 1KO. */# p,0.05. doi:10.1371/journal.pone.0053300.gRasGRP1 deficient thymi show uninterrupted cd T cell development, further suggesting that RasGRP1 is dispensable for thymic cd T cell devel.