![Figure 6. Increased microtubule stability in platelets from DIAPH1 R1213* cases. (A-C) Representative confocal microscopy images (A,C) and quantification of the microtubule surface (B) of platelets from DIAPH1 R1213* cases 10 and 16 and from a control after incubation at 4°C (A-B) or after treatment with the microtubule destabilizing toxin colchicine (10 μM; B-C). F-actin is displayed as red; posttranslationally modified α-tubulin (ac-tub and Glu-tub) as green. Values are expressed as means ± SD (n = 3 controls [black] vs case 10 and 16 [gray]; 100 platelets). Wilcoxon-Mann-Whitney test, ***P < .001. (D) Representative confocal microscopy images of platelets after spreading on fibrinogen (2.5 μg cm−2). F-actin is displayed as red; α-tubulin, ac-tub, and Glu-tub as green. Platelets in panels A, C, and D were visualized using a Leica TCS SP5 confocal microscope (Leica Microsystems). Bars represent 3 μm. (E) Quantification of the fluorescence intensity per surface unit of the immunostaining for F-actin and posttranslational modifications on α-tubulin. Values are expressed as means ± SD (n = 3 controls vs cases 10 and 16; 100 platelets). Wilcoxon-Mann-Whitney test, ***P < .001. (F) Western blots of the platelet microtubule cytoskeleton in total protein extract and in pellet or soluble fractions from a healthy control and from cases 10 and 16 separated by ultracentrifugation and probed with antibodies recognizing Tyr-tub, ac-tub, or Glu-tub. Data are presented from resting platelets and after treatment with 10 μM colchicine. Equivalent quantities of total platelet extract protein were loaded in each lane. (G-H) Densitometric analyses of the immunoblots. The data are expressed as the means ± SD of the ratios of the stable microtubule markers ac-tub (G) and Glu-tub (H) to the content of the dynamic microtubule marker Tyr-tub (n = 3 blots). Unpaired Student t test, *P < .05; **P < .01; ***P < .001. colch, 10 μM colchicine–treated platelets; α-tubulin, α-tub; F-act, F-actin; NS, nonsignificant; rest, resting platelets; P, pellet tubulin fraction; S, soluble tubulin fraction; T, total protein extract.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/127/23/10.1182_blood-2015-10-675629/4/2903f6.jpeg?Expires=1723209794&Signature=IM9c9vzn2aVvGyIys9JMSitSbkcvJ5a-2VxWxFp3jWwgmXaBXJ2MDAzPId76ve3RVritM29Yvcrt-bdfYMBAPvq0UPsWcj8l2VyOCHKtXZYlKrC309gR7ovWJqWMmAABGHMq1-GPUxRlTPNQ77TX8n9wDqP8iuRVDj8b5dZBh4sdC3-yuFNAGDq-j6u6o9vk-AL~OZoDWD7NG6~Q-uIPesWE8TOD50iDKF19~TJt6xR9-XjzUexhnMPW~BnLXpvkeTdQIQRZ903yna4j3o-rtvsABGkPTK7aqINA6KwlePwEESYhLS7xkN03T31Y3HPBk-xBL4rqpXLjwyO0UL7r9g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Increased microtubule stability in platelets from DIAPH1 R1213* cases. (A-C) Representative confocal microscopy images (A,C) and quantification of the microtubule surface (B) of platelets from DIAPH1 R1213* cases 10 and 16 and from a control after incubation at 4°C (A-B) or after treatment with the microtubule destabilizing toxin colchicine (10 μM; B-C). F-actin is displayed as red; posttranslationally modified α-tubulin (ac-tub and Glu-tub) as green. Values are expressed as means ± SD (n = 3 controls [black] vs case 10 and 16 [gray]; 100 platelets). Wilcoxon-Mann-Whitney test, ***P < .001. (D) Representative confocal microscopy images of platelets after spreading on fibrinogen (2.5 μg cm−2). F-actin is displayed as red; α-tubulin, ac-tub, and Glu-tub as green. Platelets in panels A, C, and D were visualized using a Leica TCS SP5 confocal microscope (Leica Microsystems). Bars represent 3 μm. (E) Quantification of the fluorescence intensity per surface unit of the immunostaining for F-actin and posttranslational modifications on α-tubulin. Values are expressed as means ± SD (n = 3 controls vs cases 10 and 16; 100 platelets). Wilcoxon-Mann-Whitney test, ***P < .001. (F) Western blots of the platelet microtubule cytoskeleton in total protein extract and in pellet or soluble fractions from a healthy control and from cases 10 and 16 separated by ultracentrifugation and probed with antibodies recognizing Tyr-tub, ac-tub, or Glu-tub. Data are presented from resting platelets and after treatment with 10 μM colchicine. Equivalent quantities of total platelet extract protein were loaded in each lane. (G-H) Densitometric analyses of the immunoblots. The data are expressed as the means ± SD of the ratios of the stable microtubule markers ac-tub (G) and Glu-tub (H) to the content of the dynamic microtubule marker Tyr-tub (n = 3 blots). Unpaired Student t test, *P < .05; **P < .01; ***P < .001. colch, 10 μM colchicine–treated platelets; α-tubulin, α-tub; F-act, F-actin; NS, nonsignificant; rest, resting platelets; P, pellet tubulin fraction; S, soluble tubulin fraction; T, total protein extract.
