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. 2020 Feb 14;14(4):846–864. doi: 10.1002/1878-0261.12624

Figure 2.

Figure 2

Characterization of the p27‐PAK1 interaction in OS cell lines. (A) Workflow of IP followed by mass spectrometry to identify cytoplasmic p27‐interacting proteins. (B) Results of p27 co‐IP with PAK1 in NES‐p27 cells, phosphosite mutants, and empty vector control. The pulldown assay was performed with rabbit p27 antibody (Ra‐p27), whereas PAK1 was detected by mouse PAK1 monoclonal antibody (Ma‐PAK1). Mouse monoclonal antibody p27 antibody (Ma‐p27) was used as an IP control, and total PAK1 expression was used as an input control. (C) Fluorescent images (20×) of cellular p27 and PAK1 showing p27 (EYFP, green) and PAK1 (immunofluorescence, red) proteins co‐localizing (merged, yellow) in the cytoplasm of NES‐p27 cells DAPI (blue) was used as nuclear counterstain. (D) Western blotting of phospho‐PAK1 in NES‐p27 cells, phosphosite mutants, and empty vector control. Total PAK1 protein was used as a loading control. (E) Images (left) and quantification (right) of phalloidin staining of F‐actin (stress fibers) in NES‐p27 cells, phosphosite mutants, and empty vector control (20×). For the quantification, experiments were repeated three times with ≥ 30 cells analyzed for each condition. (F) RAC1/CDC42 activity assays in NES‐p27 cells, phosphosite mutants, and empty vector control. RAC1 and CDC42 amounts of western blotting were used as input controls for RAC1/CDC42 activity assays. (G) Representative images (left) and quantification (right) of transwell migration assays of NES‐p27 and empty vector control cells treated with 1 µm of the Group I PAK inhibitor (FRAX‐597) or the vehicle control (DMSO). Migrated cells were stained, counted, and averaged using imagej software (National Institute of Mental Health, Bethesda, MD, USA) in five random and independent microscopic fields (10×). Error bars and asterisks represent standard deviations and statistical significance (Student's t‐test; *P < 0.05; **P < 0.01; ***P < 0.001, ns, not significantly, respectively). The experiment was replicated three times with a similar result. (H) Western blots showing amounts of phospho‐PAK1S144 and total PAK1 in the NES‐p27 mutant treated with FRAX‐597 or DMSO. (I) Representative images of phalloidin staining of actin stress fibers in NES‐p27 cells treated with FRAX‐597 or DMSO (40×).