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. Author manuscript; available in PMC: 2008 Mar 21.
Published in final edited form as: Eur J Immunol. 2007 Aug;37(8):2104–2116. doi: 10.1002/eji.200636680

Figure 5.

Figure 5

Inhibition of CXCL12-induced calcium mobilization and Rac activation in flotillin siRNA-transfected T cells. (A) Human primary T cells were examined 48 h post transfection with siRNA and then loaded with Fura-2 AM to evaluate [Ca2+]i mobilization in response to CXCL12 (100 ng/mL). The average fluorescence ratio for the first 60 s was standardized to a baseline value of 0, and the values are expressed as the change in the fluorescence (FL) ratio. (B) Jurkat T cells were transfected with a flotillin-1-specific siRNA or a control scrambled siRNA and then cultured for 48 h, after which the cells were treated with CXCL12 (100 ng/mL) for various times up to 30 min. At the designated times, the cells were lysed and incubated with a GST-tagged fragment of PAK PBD (p21 binding domain) protein beads (shown to bind Rac-GTP, but not Rac-GDP). Anti-Rac antibodies were utilized in the Western blot to demonstrate the extent of Rac activation at 2, 5, 15, and 30 min post CXCL12 treatment. PAK1 levels were also determined in all of these samples. This experiment was performed twice and demonstrated with similar results. (C) The densitometric intensities of the bands from (B) are shown here.