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Molecular Biology of the Cell: E03-08-0604

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Figure 1. (A) Equivalent amounts of protein from MDA-MB-231, SK-BR-3, or ZR-75-1 cells were immunoprecipitated with anti-PAK antisera and subjected to an in vitro kinase assay with histoneH3/H4 as substrate. (B) Equivalent amounts of protein from MDAMB-231, SK-BR-3, or ZR-75-1 cells were immunoprecipitated with anti-PAK antisera and subjected to Western blot analysis using anti-PAK antisera.

Figure 2. The anti-pPAK antibody specifically detects active, endogenous PAK in SKBR-3 cells. SK-BR-3 cells were infected with recombinant Semliki Forest Virus constructs encoding myc-tagged PAK1 inhibitory domain (PID) or mutant PAK inhibitory domain (PIDL107F). Cells were fixed and stained with anti-myc/ anti-mouse Alexa 568 antibodies (red) and anti-pPAK/anti-rabbit Alexa 488 (green) as described in Materials and Methods.

Figure 3. MDA-MB-231, SK-BR-3 and ZR-75-1 cells contain equivalent amounts of PIX, paxillin, NCK, and PKL. Whole cell lysates from MDA-MB-231, SK-BR-3, or ZR-75-1 cells were normalized for protein and subjected to Western blot analysis with antipaxillin, anti-PIX, anti-NCK, or anti-PKL antibodies, as described in Materials and Methods.

Figure 4. Microinjection of the PIX binding peptide reduces the area of paxillin and PIX, but not vinculin?containing focal adhesions. SK-BR-3 cells were either uninjected(control), or microinjected with the PIX binding peptide (147-231), or the Grb2 and NCK binding peptide (1-74) as described in Materials and Methods. Cells were fixed and stained with either anti-vinculin/anti-mouse Alexa 488, anti-PIX/anti-rabbit Alexa 488 or anti-paxillin/anti-mouse Alexa 488 antibodies as described in Materials and Methods. The area of PIX, paxillin or vinculin-containing focal adhesions was quantified and expressed as a percentage of total cell area using Metamorph image analysis software. Six to ten cells were analyzed for each data set. Error bars represent standard deviation for each data set. Data with an asterisk are significantly different as determined by a Student?s t-test, one-tailed, with p values <.000001.

Figure 5. Kinase-dead PAK, but not kinase-dead, GTPase binding deficient PAK abrogates pPAK staining in focal adhesions. (A) SK-BR-3 cells were infected with recombinant Semliki Forest Virus constructs encoding kinase-dead PAK (KD PAK) or kinase-dead, GTPase binding deficient PAK (KD PAK H83,86L). Cells were fixed stained with anti-pPAK/anti-rabbit Alexa 488 (green) and anti-myc/anti-mouse Alexa 568 (red). Bar = 20 μm (B) Cells were stained with anti-myc/anti-rabbit Alexa 488 and anti-paxillin/anti-mouse Alexa 568. Bar = 10 μm.

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