Figure 3: ABLIM proteins are DYRK1A substrates stabilizing F-actin.

(a) Autoradiograph of protein microarray showing duplicate spots of recombinant ABLIM1 without (left) or with (right) recombinant DYRK1A, both with radioactive ATP. (b) Co-immunoprecipitation of exogenous DYRK1A and ABLIM1 proteins in Hek293T cells. Shown is a representative blot of three independent experiments. (c) Cellular actin fractionation assay in NIH3T3 stably expressing empty vector control (mock) or ABLIM1. Jaspla, Jasplakinolide 10 nM for 30 min. Shown is a representative β-actin western blot of two independent experiments (the mean band intensity is given below). (d) Cellular actin fractionation assay in NIH3T3 stably expressing empty vector control (mock) or ABLIM2. Jaspla, Jasplakinolide 10 nM for 30 min. Shown is a representative β-actin western blot of two independent experiments (the mean band intensity is given below). (e) Deconvolution images of F-actin fibres (phalloidin) in NIH3T3 cells transiently expressing the indicated constructs. Transfected cells were identified through co-transfection of nuclear H2B–GFP. The F-actin promoting Profilin (PFN1) was included as a positive control. Scale bar, 10 μm. (f) Quantification of the microscopy experiment depicted in e (mean of two independent experiments with at least 50 cells counted in each experiment). ARR, actin-rich region (lamellipodia, stress fibres, membrane ruffles or pronounced phalloidin positivity). (g) Subcellular localization of MKL1-GFP (green) in transiently transfected Hek293A cells. Representative pictures are shown. Nuclei appear in blue (4,6-diamidino-2-phenylindole). Scale bar, 10 μm. (h) Quantification of the experiment depicted in f. C, cytoplasmic; N, nuclear; N+C, nuclear and cytoplasmic localization of MKL1-GFP (mean of n=3 independent experiments. At least 100 cells were counted). (i) SRE (serum response element) luciferase reporter assay in Hek293T cells transiently transfected with the indicated constructs (mean of n=6±s.d.). (j) Phalloidin staining of siRNA-transfected NIH3T3 cells. Shown are representative images of two independent experiments. siABlim1/2: double knockdown of Ablim1 plus Ablim2. Scale bar, 10 μm. (k) Phalloidin staining of siRNA/plasmid double-transfected NIH3T3 cells. The inset in the middle panel should help portrait the different phenotypes observed upon Ablim1 knockdown. siABlim1/2: double knockdown of Ablim1 plus Ablim2. Scale bar, 10 μm. *P<0.05; **P<0.005; ***P<0.0005 (Student’s t-test).