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. 2012 Feb 13;209(2):407–421. doi: 10.1084/jem.20111493

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© 2012 Block et al.

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Figure 6. — SLP-76 is required for the activation of Btk, PLCγ2, PI3Kγ, and p38 MAPK after E-selectin engagement. BM-derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 min, and then lysates were prepared. (A and B) Lysates of WT, Tyrobp^−/−Fcrg^−/− (A; n = 3), and Btk^−/− (B; n = 3) were immunoprecipitated (IP) with a SLP-76 antibody followed by immunoblotting (IB) with a general phosphotyrosine (PY; 4G10) antibody or total SLP-76 antibody. (C) Lysates of WT and Slp76^−/− neutrophils were immunoprecipitated with an Syk (n = 3) or Btk antibody (n = 3) followed by immunoblotting with a general phosphotyrosine (4G10) antibody, total Syk antibody (n = 3), or total Btk antibody (n = 3). Lysates were immunoblotted with antibody to phosphorylated PLCγ2 (p-PLCγ2 [Tyr1217]; n = 3), total PLCγ2 (n = 3), phosphorylated Akt (n = 3), total Akt (n = 3), phosphorylated p38 MAPK (p-p38), or total p38 MAPK (n = 3). (D) Lysates of WT and Slp76^Y112/128 neutrophils were immunoprecipitated with Btk antibody (n = 3) followed by immunoblotting with a general phosphotyrosine (4G10) antibody or total Btk antibody (n = 3). Lysates were immunoblotted with antibody to phosphorylated PLCγ2 (Tyr1217; n = 3), total PLCγ2 (n = 3), phosphorylated Akt (n = 3), total Akt (n = 3), phosphorylated p38 MAPK, or total p38 MAPK (n = 3). (E and F) Lysates of WT, Tyrobp^−/−Fcrg^−/− (E; n = 3), and Btk^−/− neutrophils (F; n = 3) were immunoprecipitated with an ADAP antibody followed by immunoblotting with a general phosphotyrosine (4G10) antibody or total ADAP antibody. (G) Lysates of WT and ADAP^−/− neutrophils were immunoprecipitated with an Syk (n = 3) or Btk antibody (n = 3) followed by immunoblotting with a general phosphotyrosine (4G10) antibody, total Syk antibody (n = 3), or total Btk antibody (n = 3). Lysates were immunoblotted with antibody to phosphorylated PLCγ2 (Tyr1217; n = 3), total PLCγ2 (n = 3), phosphorylated Akt (n = 3), total Akt (n = 3), phosphorylated p38 MAPK, or total p38 MAPK (n = 3). (H) Lysates of WT and Slp76^Y145 neutrophils were immunoprecipitated with Btk antibody followed by immunoblotting with a general phosphotyrosine antibody (4G10). Lysates were also immunoblotted with antibody to phosphorylated PLCγ2 (Tyr1217; n = 3), total PLCγ2 (n = 3), phosphorylated Akt (n = 3), total Akt (n = 3), phosphorylated p38 MAPK, or total p38 MAPK (n = 3).