Figure 6.
Impaired microtubule stability in RhoB−/− platelets. (A-B) Spreading and F-actin dynamics in RhoB−/− platelets. (A) Representative images of platelets spread on fibrinogen-coated cover slips in the presence of 0.01 U/mL thrombin for 30 minutes. (B) Quantification of platelet spreading on fibrinogen for 5, 10, and 15 minutes (n = 3). *P < .05; Mann-Whitney U test, mean plus or minus SD. (C-D) Analysis of spread platelets on fibrinogen by immunofluorescence confocal and superresolution (dSTORM) microscopy. (C) Representative confocal microscopy images of platelets spread on fibrinogen for 30 minutes with 0.01 U/mL thrombin, stained with phalloidin-Atto647 (magenta) and α-tubulin-Alexa488 (cyan), and imaged using a Leica TC SP8 confocal microscope. Scale bar in overview, 10 µm; scale bar in zoom, 5 µm (n = 4). (D) Representative dSTORM images of platelets spread on fibrinogen for 30 minutes, stained with α-tubulin-Alexa488 (red), and imaged using an in-house-made dSTORM microscope. Scale bar in overview and zoom, 5 µm (n = 2). (E-F) Analysis of cold-induced MT disassembly and reassembly of spread platelets in vitro. (E) Representative confocal immunofluorescence microscopy images of MT organization at 4°C and 37°C and a combination of disassembly at 4°C with following reassembly at 37°C in platelets. Platelets were stained with phalloidin-Atto647 (magenta) and α-tubulin-Alexa488 (cyan). Scale bar in overview, 10 µm; zoom, 3 µm (n = 3). (F) Quantification of MT organization in wt and RhoB−/− platelets after cold-induced MT disassembly and reassembly (4°C to 37°C). At least 5 images/mouse were analyzed, with minimum of 40 platelets per genotype (n = 3). *P < .05; Mann-Whitney U test, mean plus or minus SD. (G-I) Analysis of acetylation and detyrosination and polyglutaminylation of α-tubulin residues by immunoblotting. (G) Platelet and MK lysates of wt and RhoB−/− mice were immunoblotted for posttranslational modifications (PTM) of α-tubulin(acetylated α-tubulin, detyrosinated α-tubulin, and polyglutaminylated α-tubulin). (H-I) Quantification of PTM immunoblots. Each data point represents 1 mouse (acetylated α-tubulin n = 7; glutaminylated α-tubulin n = 5). *P < .05; Mann-Whitney U test, mean plus or minus SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TB, tubulin.

Impaired microtubule stability in RhoB−/− platelets. (A-B) Spreading and F-actin dynamics in RhoB−/− platelets. (A) Representative images of platelets spread on fibrinogen-coated cover slips in the presence of 0.01 U/mL thrombin for 30 minutes. (B) Quantification of platelet spreading on fibrinogen for 5, 10, and 15 minutes (n = 3). *P < .05; Mann-Whitney U test, mean plus or minus SD. (C-D) Analysis of spread platelets on fibrinogen by immunofluorescence confocal and superresolution (dSTORM) microscopy. (C) Representative confocal microscopy images of platelets spread on fibrinogen for 30 minutes with 0.01 U/mL thrombin, stained with phalloidin-Atto647 (magenta) and α-tubulin-Alexa488 (cyan), and imaged using a Leica TC SP8 confocal microscope. Scale bar in overview, 10 µm; scale bar in zoom, 5 µm (n = 4). (D) Representative dSTORM images of platelets spread on fibrinogen for 30 minutes, stained with α-tubulin-Alexa488 (red), and imaged using an in-house-made dSTORM microscope. Scale bar in overview and zoom, 5 µm (n = 2). (E-F) Analysis of cold-induced MT disassembly and reassembly of spread platelets in vitro. (E) Representative confocal immunofluorescence microscopy images of MT organization at 4°C and 37°C and a combination of disassembly at 4°C with following reassembly at 37°C in platelets. Platelets were stained with phalloidin-Atto647 (magenta) and α-tubulin-Alexa488 (cyan). Scale bar in overview, 10 µm; zoom, 3 µm (n = 3). (F) Quantification of MT organization in wt and RhoB−/− platelets after cold-induced MT disassembly and reassembly (4°C to 37°C). At least 5 images/mouse were analyzed, with minimum of 40 platelets per genotype (n = 3). *P < .05; Mann-Whitney U test, mean plus or minus SD. (G-I) Analysis of acetylation and detyrosination and polyglutaminylation of α-tubulin residues by immunoblotting. (G) Platelet and MK lysates of wt and RhoB−/− mice were immunoblotted for posttranslational modifications (PTM) of α-tubulin(acetylated α-tubulin, detyrosinated α-tubulin, and polyglutaminylated α-tubulin). (H-I) Quantification of PTM immunoblots. Each data point represents 1 mouse (acetylated α-tubulin n = 7; glutaminylated α-tubulin n = 5). *P < .05; Mann-Whitney U test, mean plus or minus SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TB, tubulin.

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