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. 2021 Mar 27;17(5):1315–1327. doi: 10.7150/ijbs.58102

Figure 4.

Figure 4

PAK5 inhibits apoptosis by blocking AIF nuclear translocation. (a) PAK5 directly binds to AIF amino acids 263 and 480. Black stars indicate GST and GST-fusion proteins. (b,c) Separation of nucleus and cytoplasm experiment showing that PAK5 expression decreased the nuclear translocation of AIF. The data are shown as the mean _ SEM of triplicate experiments (***p < 0.001, ****p < 0.0001 vs. Control, n =3). (d,e) PAK5 and AIF interact in the cytoplasm. Separation of cytoplasmic protein and nuclear protein with nucleoplasm separation Kit, lysates were subjected to immunoprecipitation and western blot with antibodies as indicated. (f) The interaction of AIF with importins was determined by immunoprecipitation assay. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (g) Overexpression of PAK5 diminished AIF interaction with importin α3. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (h) Knockdown of PAK5 enhanced AIF interaction with importin α3. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (i,j) Overexpression(i)/knockdown(j) of PAK5 had no effect on AIF interaction with importin α3. Lysates were immunoprecipitated with anti-GST antibody and immunoblotted with indicated antibodies. (k) PAK5 can inhibit apoptosis of breast cancer cells.BT474 cells transfected with the indicated constructs. The apoptosis was detected by flow cytometry. On the two-dimensional scatter diagram, PI and annexin V are x and Y axes respectively, the second quadrant represents apoptotic cells, and the fourth quadrant represents early apoptotic cells. The data are presented as a histogram of the mean _ SEM of three independent experiments (**p < 0.01, ***p < 0.001, n =3).