Dynamic changes of tubulin acetylation in activated platelets. (A) Human platelets were fixed after different spreading periods (as indicated, ON indicates overnight) on glass surfaces and stained using an antiacetylated tubulin antibody (clone 6-11B-1, Sigma-Aldrich, T6793; green, top panels) or an antitubulin antibody (clone B-5-1-2, Sigma-Aldrich, T5168; green, bottom panels) detected with AlexaFluor-488 goat anti–mouse IgGs (Invitrogen; A11029). Colabeling of the actin cytoskeleton with phalloidin-rhodamine (Sigma-Aldrich, P1951; red) served as an indicator for the extent of platelet spreading. Fluorescent images were acquired using an upright microscope (Olympus BX41) equipped with a color camera DP70 using a 100× oil immersion objective and the software analySIS. (B) Human platelets were incubated for 40 minutes (left panels) or 9 hours (right panels) on glass cover slips coated with 20 μg/mL collagen (Col), fibrinogen (Fg), fibronectin (Fn), or poly-L-lysine (PLL) and blocked with 3% BSA. Platelets were then fixed and stained using an antiacetylated tubulin antibody and phalloidin-rhodamine for the actin cytoskeleton. (C) Platelets in human PRP were induced or not (no) to aggregate with 1.5mM arachidonic acid (AA), 10μM adenosine diphosphate (ADP), or 10 μg/mL collagen (Col). Aggregation was followed using an APACT 4004/LABiTec aggregometer. The PRP was then centrifuged and the platelet pellet lysed and analyzed by Western blot using an acetylated tubulin and an antitubulin antibody (inset). (D) Human platelets were allowed to spread on glass Petri dishes (6 × 10⁷/10 mL/dish) for the indicated periods of time and then scrapped and analyzed by Western blot using an acetylated tubulin antibody. The same membrane was stained with Coomassie as a loading control. (E) Human PRP was incubated for 20 minutes on ice or at room temperature and then centrifuged. The platelet pellet was lysed and analyzed by Western blot using an acetylated tubulin and an antitubulin antibody. (F) Human platelets were incubated for 30 minutes at room temperature with 15 μg/mL nocodazole, 25μM taxol, or without drug and then either fixed in suspension (top panel) or allowed to spread on glass coverslips for 60 minutes (bottom panel). Platelets were then stained with the mouse monoclonal antiacetylated tubulin antibody and a monoclonal rabbit antitubulin antibody (clone EP1332Y, Millipore, 04-1117) detected with AlexaFluor-546 goat antirabbit IgGs (Invitrogen; A11035) as indicated. Scale bars represent 10 μm.