Figure 4.
Figure 4. Expression and activity of group I PAKs in ALL. (A) Immunoblot analysis of PAK2 S141 phosphorylation in XT-ALL22089 and SEM cells after FLT3L stimulation or pharmacological inhibition of FLT3 with PKC412 or CEP-701 compared with solvent control. PAK2 S141 phosphorylation was observed in additional FLT3-expressing XT-ALLs and ALL cell lines (supplemental Figure 7G-H). β-actin served as loading control. (B) Immunoprecipitation (IP) of PAK1 vs PAK2 and phosphoimmunoblot analysis of pT402/432 in catalytic domains of PAK2 and PAK1, respectively. SEM cells were stimulated with FLT3L and harvested at indicated times. Pull-down efficiencies were determined by PAK1/PAK2–directed immunoblot analysis. Input control using approximately one-tenth of protein lysate. Arrowheads mark specific PAK isoforms. (C) Growth factor (FLT3L, PDGF-BB, IGF-1) stimulation leads to activation of PAK1 and PAK2 in SEM cells as demonstrated by IVK assay using H4 as substrate. (D) IVK assay after PAK1 or PAK2 IP in FLT3L-stimulated MHH-CALL2 cells treated with CDC42 inhibitor ML141, RAC1 inhibitor NSC23766, and FAK inhibitor PF-573228 at indicated concentrations compared with nonstimulated, untreated control. (E) Phosphoimmunoblot of growth factor induced canonical signaling pathways including MAPK, AKT, S6 kinase, GSK3β, STAT3, and STAT5 in 3 distinct XT-ALLs. β-actin served as loading control. (F) Phosphoimmunoblot of STAT5 activation (pY694) in various ALL models upon FLT3L stimulation. Total STAT5a/b served as loading control. (G) Mean relative expression plus or minus SD of PAK1 and PAK2 transcripts in primary B-cell precursor ALLs (n = 122) was determined by RT-PCR normalized toward β2-microglobulin. (H) In analogy to panel G, mean relative expression of PAK1 and PAK2 transcripts plus or minus SD in HSCs, lymphopoietic precursors (CLP, pro-B, pre-B), mature peripheral blood CD19+ B cells, and CD3+ T cells. CD34+ hematopoietic stem/precursor cells were purified from umbilical cord blood (refer also to supplemental Figure 1D). mRNA, messenger RNA.

Expression and activity of group I PAKs in ALL. (A) Immunoblot analysis of PAK2 S141 phosphorylation in XT-ALL22089 and SEM cells after FLT3L stimulation or pharmacological inhibition of FLT3 with PKC412 or CEP-701 compared with solvent control. PAK2 S141 phosphorylation was observed in additional FLT3-expressing XT-ALLs and ALL cell lines (supplemental Figure 7G-H). β-actin served as loading control. (B) Immunoprecipitation (IP) of PAK1 vs PAK2 and phosphoimmunoblot analysis of pT402/432 in catalytic domains of PAK2 and PAK1, respectively. SEM cells were stimulated with FLT3L and harvested at indicated times. Pull-down efficiencies were determined by PAK1/PAK2–directed immunoblot analysis. Input control using approximately one-tenth of protein lysate. Arrowheads mark specific PAK isoforms. (C) Growth factor (FLT3L, PDGF-BB, IGF-1) stimulation leads to activation of PAK1 and PAK2 in SEM cells as demonstrated by IVK assay using H4 as substrate. (D) IVK assay after PAK1 or PAK2 IP in FLT3L-stimulated MHH-CALL2 cells treated with CDC42 inhibitor ML141, RAC1 inhibitor NSC23766, and FAK inhibitor PF-573228 at indicated concentrations compared with nonstimulated, untreated control. (E) Phosphoimmunoblot of growth factor induced canonical signaling pathways including MAPK, AKT, S6 kinase, GSK3β, STAT3, and STAT5 in 3 distinct XT-ALLs. β-actin served as loading control. (F) Phosphoimmunoblot of STAT5 activation (pY694) in various ALL models upon FLT3L stimulation. Total STAT5a/b served as loading control. (G) Mean relative expression plus or minus SD of PAK1 and PAK2 transcripts in primary B-cell precursor ALLs (n = 122) was determined by RT-PCR normalized toward β2-microglobulin. (H) In analogy to panel G, mean relative expression of PAK1 and PAK2 transcripts plus or minus SD in HSCs, lymphopoietic precursors (CLP, pro-B, pre-B), mature peripheral blood CD19+ B cells, and CD3+ T cells. CD34+ hematopoietic stem/precursor cells were purified from umbilical cord blood (refer also to supplemental Figure 1D). mRNA, messenger RNA.

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