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Fig. 1. PAK1 regulates CSF1-induced MAPK activation but not macrophage differentiation or chemotaxis. (A) Lysates from WT and PAK1^–/– BMMs were immunoblotted for PAK1 and PAK2 using a group-1-specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was detected as a loading control. Gr, cells in growth medium. (B) Flow cytometry analysis of WT and PAK1^–/– BMM surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. (C) WT BMMs were stimulated with 33 ng/ml CSF1, and lysates were immunoblotted for Thr423-P-PAK1 (P-PAK1) and β-actin as a loading control. (D) WT and PAK1^–/– BMMs were stimulated with 33 ng/ml CSF1 and lysates were immunoblotted for Ser473-P-Akt, Thr202/Tyr204-P-ERK1/2, Thr180/Tyr182-P-p38 and Ser298-P-MEK1/2 levels. β-actin was detected as a loading control. Western blots are representative of three separate experiments. (E) To investigate chemotaxis, 1x10⁵ WT or PAK1^–/– BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean ± s.e.m. of three experiments performed in triplicate.

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