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Fig. 4. PAK1 promotes ERK1/2 activation at the cell periphery. (A) WT and PAK1–/– BMMs were adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for Thr202/Tyr204-P-ERK1/2, Thr180/Tyr182-P-p38, Ser298-P-MEK1/2 and total ERK1/2. Densitometry quantification of phosphorylated ERK1/2 and p38 levels equalised to total ERK1/2 protein levels are shown (a.u., arbitrary units). Data are mean ± s.d. of two separate experiments. (B) WT and PAK1–/– BMMs were plated onto glass coverslips for 10 minutes in growth medium and were stained using an ERK1/2 antibody and TRITC-phalloidin to visualise F-actin. Cells were imaged by confocal microscopy. ERK1/2 localisation was quantified by determining the number of cells with ERK1/2 staining at the periphery. The mean ± s.d. is shown for two separate experiments; n=60 (WT) and n=45 (PAK1–/–). (C) BMMs were stained with a Thr202/Tyr204-P-ERK1/2 antibody (P-ERK1/2) and TRITC-phalloidin (F-actin). Localisation of ERK1/2-P was quantified by determining the number of cells with staining at the cell periphery. The mean ± s.d. is shown for two separate experiments; n=39 (WT) and n=62 (PAK1–/–). (D) WT and PAK1–/– BMMs were kept in suspension or adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for Ser217/221-P-MEK1/2, and total ERK1/2 as a loading control. Densitometry quantification of phosphorylated MEK1/2 equalised to total ERK1/2 protein levels is shown (a.u., arbitrary units). Data are mean ± s.d. of two separate experiments.
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