Figure 3
Figure 3. Effect of CD9 expression on Aggrus-induced platelet aggregation. (A) HT1080 cells that had been stably transfected with expression vector encoding nothing, V5-tagged CD9, or V5-tagged CD44 were lysed and immunoblotted with antibodies to Aggrus, V5 tag, or β-actin. (B) HT1080 cells that had been transfected with nothing (black line), control siRNA (red line), or aggrus-specific siRNA (green line) were incubated with mouse PRP. Then, light transmittance was monitored for 15 minutes. The blue arrow indicates when cells are added. Aggrus expression level in each sample was estimated by Western blot analysis with an antibody to Aggrus or β-actin (right panels). (C) HT1080 cells were preincubated with control rat IgG (black line) or rat anti-Aggrus antibody (red line). Then, the reactions were incubated with mouse PRP. Light transmittance was monitored for 15 minutes. (D) Stably transfected polyclonal HT1080/mock (black line), HT1080/CD9 (red line), and HT1080/CD44 (green line) were incubated with mouse platelet-rich plasma. Then, light transmittance was monitored for 15 minutes (left panel). The blue arrow indicates when cells are added; red arrow, when platelet aggregation begins. Platelet aggregation–inducing activity was calculated using the following formula: platelet aggregation-inducing activity = 1 / A, where A is the time from reaction-starting point to maximum value point. Each point represents a mean plus or minus SD of triplicate experiments. *P < .05; **P < .005 using the Student t test. (E) Stably transfected polyclonal HT1080/mock and HT1080/CD9 cells were added to platelets, and reactions were terminated by the addition of 2 × lysis buffer after indicated times. The lysates were electrophoresed and immunoblotted with an antibody to Erk1/2 and phospho-Erk1/2. (F) CHO cells were transiently transfected with a CD9-expressing vector (CHO/CD9; red line) or an empty vector (CHO/mock; black line). Mouse PRP was preincubated with CHO/mock or CHO/CD9 for 10 minutes. Then, 10 μL of ADP (40 μM in PBS; left panel) or thrombin (50 U/mL in PBS; right panel) was added to the reactions.

Effect of CD9 expression on Aggrus-induced platelet aggregation. (A) HT1080 cells that had been stably transfected with expression vector encoding nothing, V5-tagged CD9, or V5-tagged CD44 were lysed and immunoblotted with antibodies to Aggrus, V5 tag, or β-actin. (B) HT1080 cells that had been transfected with nothing (black line), control siRNA (red line), or aggrus-specific siRNA (green line) were incubated with mouse PRP. Then, light transmittance was monitored for 15 minutes. The blue arrow indicates when cells are added. Aggrus expression level in each sample was estimated by Western blot analysis with an antibody to Aggrus or β-actin (right panels). (C) HT1080 cells were preincubated with control rat IgG (black line) or rat anti-Aggrus antibody (red line). Then, the reactions were incubated with mouse PRP. Light transmittance was monitored for 15 minutes. (D) Stably transfected polyclonal HT1080/mock (black line), HT1080/CD9 (red line), and HT1080/CD44 (green line) were incubated with mouse platelet-rich plasma. Then, light transmittance was monitored for 15 minutes (left panel). The blue arrow indicates when cells are added; red arrow, when platelet aggregation begins. Platelet aggregation–inducing activity was calculated using the following formula: platelet aggregation-inducing activity = 1 / A, where A is the time from reaction-starting point to maximum value point. Each point represents a mean plus or minus SD of triplicate experiments. *P < .05; **P < .005 using the Student t test. (E) Stably transfected polyclonal HT1080/mock and HT1080/CD9 cells were added to platelets, and reactions were terminated by the addition of 2 × lysis buffer after indicated times. The lysates were electrophoresed and immunoblotted with an antibody to Erk1/2 and phospho-Erk1/2. (F) CHO cells were transiently transfected with a CD9-expressing vector (CHO/CD9; red line) or an empty vector (CHO/mock; black line). Mouse PRP was preincubated with CHO/mock or CHO/CD9 for 10 minutes. Then, 10 μL of ADP (40 μM in PBS; left panel) or thrombin (50 U/mL in PBS; right panel) was added to the reactions.

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