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. 2015 Dec 4;6:8982. doi: 10.1038/ncomms9982

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(a) Control or Rap1KD cells stimulated with CXCL12 as indicated. Cell lysates were probed with antibody against ERM phosphorylated at Thr 567 and 558, or anti-ERM. (b; Left) Lysates from BAF cells transfected with vector (control) or Spa-1 immunoblotted with anti-Spa-1. Actin is a loading control. (Right upper) Cells stimulated with CXCL12 as indicated. Cell lysates were pulled down with GST-RalRBD, and immunoblotted with anti-Rap1. (Right lower) Control or Spa-1-expressing cells were stimulated with CXCL12 as indicated. Cell lysates were probed with anti-phosphorylated ERM, or anti-ERM. (c) Phosphorylation of ERM in f/f or −/− CD4⁺ T_N cells. T_N cells were stimulated with CCL21 as indicated. Total lysates from f/f or −/− T_N cells were probed with antibody against phosphorylated ERM or anti-ERM. Actin is a loading control. (d) Phosphorylation of ERM in f/f or −/− CD4⁺ T_EM cells. T_EM cells were stimulated with CXCL10 for 15 s. Total lysates from f/f or −/− T cells were probed with antibody against phosphorylated ERM, or anti-ERM. (e) Control or Rap1KD cells were stimulated with CXCL12 as indicated. Cell lysates were pulled down with GST-Rhotekin RBD and immunoblotted with an anti-Rho antibody. (f) COS cells were transfected with FLAG-LOK, together with T7-tagged Rap1V12 or Rap1N17. (Left) Cell lysates were immunoprecipitated with anti-T7 and immunoblotted with an anti-FLAG antibody. (Right) Kinase activity of LOK in Rap1N17 or V12-expressing COS cells measured using Myelin basic protein (MBP) as the substrate. Total FLAG-LOK or T7-Rap1 was immunoblotted with an anti-FLAG or T7 antibody. (g; Left upper) Control or Rap1KD cells transfected with FLAG-LOK were stimulated with CXCL12 as indicated. Kinase activity of LOK was measured. Total FLAG-LOK was immunoblotted with an anti-FLAG antibody. (Left lower) Kinase activity of LOK in f/f or −/− T_EM cells stimulated with CXCL10 for 15 s. Total LOK was immunoblotted with an anti-LOK antibody. (Right) Kinase activity of LOK in control or Spa-1-expressing cells stimulated with CXCL12 as indicated. Total FLAG-LOK was immunoblotted with an anti-FLAG antibody. For all blots, a representative of three or four independent experiments is shown.