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Fig. 9. DAP-kinase-induced phosphorylation of Ser283 within tropomyosin-1 promotes actin stress fiber formation. (A) Exponentially growing HUVECs were plated in gelatin-coated Petri dishes after being co-electroporated without (a-d) or with (e-p) a siRNA for DAP kinase together with or without (a-h) a plasmid expressing either tropomyosin-1 mutant S283A (i-l) or S283E (m-p). A plasmid expressing GFP was co-electroporated in each condition as a marker of positive cells. The next day, cells were treated (c,d,g,h,k,l,o,p) or not (a,b,e,f,i,j,m,n) with H₂O₂ (250 µM) for 30 minutes. Thereafter, cells were fixed and stained for F-actin using Alexa-488-phalloidin (b,c,f,g,j,k,n,o) and for GFP using a rabbit polyclonal antibody against GFP, revealed with anti-rabbit-IgG Alexa 568 (a,d,e,h,i,l,m,p), and then were examined by confocal microscopy. (B) A quantification from the above experiments was performed. GFP-positive cells expressing transcytoplasmic actin stress fibers or membrane blebs were counted and the ratio over the total number of counted GFP-positive cells was calculated from two separated experiments. Results are representative of means ± s.e.m. of at least three experiments. (C) Extracts of the cells from the above experiment were made and were processed for immunodetection of DAP kinase (DAPK; upper panel) and actin (lower panel) using specific antibodies. C, control cells; D, DAPK siRNA cells; A, DAPK siRNA + TM-1 S283A cells; E, DAPK siRNA + TM-1 S283E cells.

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