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. 2006 Apr;26(8):3215–3230. doi: 10.1128/MCB.26.8.3215-3230.2006

FIG. 2.

FIG. 2.

Biochemical characterization of Pak5. (A) CHO cells were cotransfected with equal amounts of expression vectors encoding HA-tagged Cdc42 L61 and Myc-tagged wt-Pak5 or mut-Pak5 or with an empty vector. After transient expression, Myc-wt-Pak5 and Myc-mut-Pak5 were immunoprecipitated from total lysates with anti-Myc antibodies and protein A-agarose. Immunoblotting was then performed, and the complexes were revealed with anti-HA antibodies. The bottom panel shows the expression of Pak5, revealed with anti-Myc antibodies, and Cdc42, revealed with anti-HA antibodies, from extracts of WCL. (B) CHO cells were transfected with equal amounts of expression vectors encoding Myc-tagged wt-Pak5 and mut-Pak5 or with an empty vector. After transient expression, Myc-Pak5 proteins were pulled down from WCL with GST-Cdc42 loaded with GTP. Immunoblotting was then performed using anti-Myc and anti-GST antibodies. The expression level of Pak5 in the lysates used for the pull-down assay is shown in the bottom panel (expression was detected by immunoblotting using anti-Myc antibodies). (C) CHO cells were transfected with expression vectors for Myc-tagged wt- or mut-Pak5. Proteins were immunoprecipitated with an anti-Myc antibody, and a kinase assay was performed as described in Materials and Methods, using [γ-32P]ATP and myelin basic protein (MBP) as a substrate. Reaction products were separated by SDS-PAGE and subjected to autoradiography. The bottom panel shows the expression levels of proteins in the WCL prior to immunoprecipitation. (D) CHO cells were transfected with expression vectors for Myc-tagged wt- or mut-Pak5. Proteins were immunoprecipitated with an anti-Myc antibody, and a kinase assay was performed as described in Materials and Methods, using His-tagged recombinant BAD as a substrate. Reaction products were separated by SDS-PAGE and subjected to immunoblotting using antiphosphoserine antibodies. The bottom panels show the expression levels of proteins in the WCL prior to immunoprecipitation and the amount of His-BAD added to each reaction.