Fig 2: Inhibition of IL-31-induced neurite outgrowth by FRAX597.Neuro2A cells were treated with anti-IL-31Ra antibody in the absence or presence of FRAX597 at indicated concentrations. Phosphorylated PAK2 at Ser141 were analyzed by immunoblotting (A). The representative images are shown on the left, and the quantitative data of the ratios of phosphorylated PAK2 versus PAK2 are shown on the right. Neurites were defined as a process with lengths equivalent to one diameter of a cell body. The percentage of neurite-bearing cells was calculated from the total number of counted cells. The representative micrographs are shown (B), and the quantitative data of the neurite outgrowth are shown (C).Scale bar: 20 µm. Data are pooled from three independent experiments and shown as mean and SD. **p < 0.01 as compared with control; ##p < 0.01, #p < 0.05 as compared with anti-IL-31Ra antibody-treated group (one-way ANOVA/Tukey-Kramer post-hoc comparisons).

Fig 3: PAK2 knockdown decreases colony formation. (A) Colony formation assays of KU812 cells expressing shRDM, shPAK1, or shPAK2 (n = 4). (B) Quantification of colonies depicted in (A). Graphs represent means ± SEM. (C) Expression of PAK1 and PAK2 in single shPAK2+ colonies. Rel. expr. comp.: Relative expression compared.

Fig 4: STAT5S725 and STAT5S779 phosphorylations are independent of BCR-ABLp185 kinase activity and STAT5Y694 phosphorylation. (a) BCR-ABLp185+ cells were treated with imatinib (2 µM) and STAT5 phosphorylation on residues Y694, S725 and S779 monitored by immunoblotting. (b) Immunoblotting of K562 cells after treatment with 2 µM imatinib for indicated time points. Levels of active versions of PAK1 and PAK2 (pPAK1T423; pPAK2T402) do not alter upon inhibition of BCR-ABL kinase activity. (c) Immunoblotting of Stat5fl/fl Mx-1Cre+ BCR-ABLp185+ cells expressing indicated STAT5 mutants. Endogenous Stat5 was deleted via interferon-ß (IFN-ß) treatment 4 weeks before use. STAT5 proteins harboring a Y694 mutation maintain phosphorylations on S725 and S779. (d) Dose–response curves of PAK2 shRNA- or random shRNA-expressing BCR-ABLp185+ cells toward PF-3758309 (left panel) or imatinib (right panel). (e) Scheme summarizing activation of STAT5 via PAK1/2 kinases independently of BCR-ABL kinase activity.

Fig 5: PAK is necessary for PDGF but not for EGF-mediated AKT Ser473 phosphorylation whereas RICTOR knockdown inhibits both pathways.(A) PAK1 and PAK2 expression was suppressed in MCF7 cells using siRNAs and the cells were stimulated with 20 ng/ml PDGF-BB for 10 min. A representative western blot is shown and AKT pSer473 is quantified below (mean ± s.e.m.; n = 2, ** represents p<0.05). (B) MCF7 and HeLa cells were transfected as above and stimulated with 20 ng/ml EGF. A representative western blot is shown and below quantification of AKT pSer473 in MCF7 and HeLa cells (mean ± s.e.m.; n = 3) is presented. (C) HeLa cells were treated with PAK inhibitor IPA3 (30 µM) or DMSO as vehicle control and then stimulated with 20 ng/ml PDGF-BB or EGF. A representative western blot is shown. The graph provides quantification of AKT pSer473 levels, after EGF-stimulation of cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in EGF-stimulated cells without the inhibitor (mean ± s.e.m.; n = 3). (D) RICTOR expression was suppressed in HeLa cells using siRNA and the cells were stimulated with EGF (20 ng/ml). Cell lysates were subjected to SDS-PAGE followed by western blotting using antibodies as indicated. A representative blot is shown and quantification of AKT pS473 levels is found below (mean ± s.e.m.; n = 3, ** represents p<0.05). (E) HeLa cells transfected with RICTOR-directed siRNA were stimulated with 20 ng/ml PDGF-BB and analysed as in (D). A representative western blot is shown and the graph below shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2, * represents p<0.005). (F) HeLa cells, transfected with non-target or ILK-directed siRNA, were stimulated with 20 ng/ml EGF and the samples analysed as explained above. The graph shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2).