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. 2009 Jun 1;29(15):4156–4166. doi: 10.1128/MCB.01813-08

FIG. 6.

FIG. 6.

PAK1 acts downstream of Rac1 in the release of transcriptional repression by BCL-6. (A) DLD-1 cells were transfected with the transcriptional BCL-6 luciferase reporter vector and one of the indicated GFP-tagged expression vectors encoding either activated small GTPase mutants or protein kinase mutants. Luciferase activity was determined in cell lysates, and expression of transfected proteins was documented by Western blotting. A graph with the observed changes in luciferase activity relative to GFP empty vector-transfected control cells (top panel) and immunoblots with the expression levels of the GFP-tagged proteins (middle panel) and β-tubulin as a loading control (bottom panel) are shown. The migration of molecular size markers is indicated. Note that transcriptional repression by BCL-6 was released in the presence of Rac1-L61, Cdc42-V12, and a constitutively active (CA) PAK1 mutant, whereas a kinase-dead (KD) PAK1 prevented the Rac1-L61 mediated increase in luciferase activity. α-GFP, anti-GFP antibody. Symbols: +, transfected or treated with drug; −, not transfected or treated with drug. (B) RT-PCR analysis to determine the expression of PAK1, PAK2, and PAK3 in DLD-1 and HT29 colorectal cells compared to SW1088 glioblastoma cells. (C) Western blot analysis to directly compare the expression levels of PAK1 and PAK2 in DLD-1 or HT29 cells using an anti-PAK1/2/3 antibody. Note that PAK1 is the most prominent isoform expressed.