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. 2015 Jan 21;6(6):4345–4356. doi: 10.18632/oncotarget.2999

Figure 3. GATA1 is a physiological substrate of p21-activated kinase 5.

Figure 3

(A) Western blot analysis demonstrates the protein level of PAK5 and GATA1 in breast cancer tissues and matched adjacent noncancerous tissues. N, matched adjacent noncancerous tissue; T, breast cancer tissue. **p < 0.01. (B) PAK5wt/KM and GATA1 were transfected into MCF-7 cells as indicated for Luciferase Assays. *p < 0.05, **p < 0.01. (C) HEK-293 cells transfected with Myc-PAK5KD (PAK5 kinase domain) were lysed for IP with anti-Myc antibody, the immunoprecipitated PAK5 kinase was incubated with GST or GST-GATA1 for in vitro kinase assay. Histone H3 (HH3) served as a positive control. (D) In vitro kinase assay using commercial PAK5 kinase and GST-GATA1FL (Full length, 1–413aa) or GST-GATA1 deletion mutants (1–160aa, 1–300aa and 241–413aa). Histone H3 (HH3) served as a positive control. (E) In vitro kinase assay using commercial PAK5 kinase and GST-GATA1 wt or GST-GATA1 single-site mutations as indicated. (F) MCF-7 cells transfected with GATA1 wt/S161A S187A and PAK5 wt were used for Ser/Thr phosphoprotein purification. Then concentrated protein was used for western blot. The top lane, the phosphorylated GATA1 from these cells lysates was used for immunoblotting using anti-GFP antibody. Total cells lysates were used for immunoblotting by anti-GFP, GAPDH antibodies.