Journal: Blood Advances

Article Title: Calpain cleaves phospholipid flippase ATP8A1 during apoptosis in platelets

doi: 10.1182/bloodadvances.2018023473

Figure Lengend Snippet: Calpain-mediated cleavage of ATP8A1 depends on caspase activation and calcium influx during platelet apoptosis. (A) Washed mouse platelets (5 × 107) were treated with vehicle (DMSO) or ABT737 (1 µM, 37°C) in the absence or presence of CaCl2 (2 mM) for the indicated time. In some experiments, platelets were preincubated with DMSO (DS), CP (50 µg/mL), or QVD (25 µM) at room temperature for 15 minutes before treatment with ABT737 (2 hours). Platelets were subsequently lysed for western blot analysis of ATP8A1, Caspase-3, Calpain-1, and β-actin. (B) Washed mouse platelets (5 × 107) were preloaded with Fluo-4 and treated with vehicle (DMSO) or ABT737 (1 µM) and CaCl2 (1.3 mM) at 37°C for 2 hours in the absence or presence of CP (50 µg/mL) or QVD (25 µM). Fluo-4 fluorescence was measured, and data are expressed as mean fluorescence intensity (MFI) ± SEM (n = 3). Immunoblots are representative of 3 independent experiments. Bar graphs represent blot quantification of full-length ATP8A1 by densitometric analysis (means ± SEM, n = 3) of stained bands using Image J, corrected for loading control (β-Actin). Statistical significance was calculated using Student 2-tailed t test. **P < .01; ***P < .001.

Article Snippet: Membranes were probed with primary antibodies against ATP8A1 (1:1000; Proteintech), ATP11C (1:1000; customized, Pineda Antikörper-Service), caspase-3 (1:1000; Cell Signaling), calpain-1 large subunit (1:1000; Cell Signaling), gelsolin (1:1000; Abcam), β-actin (1:2000; Abcam), or Na + /K + -ATPase (Clone EP1845Y, 1:5000; Abcam) in PBS-Tween 20 with 2% membrane blocking reagent (GE Healthcare) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (1:2000; GE Healthcare) at room temperature for 1 hour.

Techniques: Activation Assay, Western Blot, Fluorescence, Staining

Journal: Blood Advances

Article Title: Calpain cleaves phospholipid flippase ATP8A1 during apoptosis in platelets

doi: 10.1182/bloodadvances.2018023473

Figure Lengend Snippet: ATP8A1 is a direct substrate of calpain. (A) Mouse platelet membrane fractions (10 µg) remained untreated or were incubated with purified human calpain-1 (4U) in the absence or presence of CaCl2 (5 mM) or CP (50 µg/mL) at room temperature for 30 minutes. Samples were then denatured for western blot analysis of ATP8A1 and β-actin. (B-C) Washed mouse platelets (5 × 107) were treated with vehicle (DMSO) or A23187 (1 µM; room temperature) in the presence of CaCl2 (2 mM) for the indicated time. In some experiments, platelets were preincubated with DMSO (DS), CP (50 µg/mL) or QVD (25 µM) at room temperature for 15 minutes before treatment with A23187 (20 minutes) or ABT737 (1 µM, 37°C, 2 hours, in the absence of CaCl2). Platelets were subsequently lysed for western blot analysis of ATP8A1, Caspase-3, Calpain-1, Gelsolin, and β-actin. Immunoblots are representative of n = 3 independent experiments. Bar graphs represent blot quantification of full-length ATP8A1 by densitometric analysis (means ± SEM, n = 3) of stained bands using Image J, corrected for loading control (β-Actin). Statistical significance was calculated using Student 2-tailed t test. *P < .05; **P < .01; ***P < .001.

Article Snippet: Membranes were probed with primary antibodies against ATP8A1 (1:1000; Proteintech), ATP11C (1:1000; customized, Pineda Antikörper-Service), caspase-3 (1:1000; Cell Signaling), calpain-1 large subunit (1:1000; Cell Signaling), gelsolin (1:1000; Abcam), β-actin (1:2000; Abcam), or Na + /K + -ATPase (Clone EP1845Y, 1:5000; Abcam) in PBS-Tween 20 with 2% membrane blocking reagent (GE Healthcare) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (1:2000; GE Healthcare) at room temperature for 1 hour.

Techniques: Incubation, Purification, Western Blot, Staining

Journal: Blood Advances

Article Title: Calpain cleaves phospholipid flippase ATP8A1 during apoptosis in platelets

doi: 10.1182/bloodadvances.2018023473

Figure Lengend Snippet: ATP8A1 is not cleaved in platelets activated by physiological agonists. (A-B, upper panels) Bar graphs represent means ± SEM (n = 3) of the percentage of Annexin-V-positive cells in platelets stimulated by A23187, thrombin, collagen, or thrombin and collagen in the presence of CaCl2 (2 mM) at room temperature for 20 minutes. (A-B, lower panels) Washed mouse platelets (5 × 107) were treated with vehicle, A23187, thrombin, collagen, or thrombin and collagen in the presence of CaCl2 (2 mM) at room temperature for 20 minutes. Platelets were subsequently lysed for western blot analysis of ATP8A1, Caspase-3, Calpain-1, and β-actin. Immunoblots are representative of 3 independent experiments. Bar graphs represent blot quantification of full-length ATP8A1 by densitometric analysis (means ± SEM, n = 3) of stained bands using Image J, corrected for loading control (β-Actin). Statistical significance was calculated using Student 2-tailed t test. ***P < .001.

Article Snippet: Membranes were probed with primary antibodies against ATP8A1 (1:1000; Proteintech), ATP11C (1:1000; customized, Pineda Antikörper-Service), caspase-3 (1:1000; Cell Signaling), calpain-1 large subunit (1:1000; Cell Signaling), gelsolin (1:1000; Abcam), β-actin (1:2000; Abcam), or Na + /K + -ATPase (Clone EP1845Y, 1:5000; Abcam) in PBS-Tween 20 with 2% membrane blocking reagent (GE Healthcare) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (1:2000; GE Healthcare) at room temperature for 1 hour.

Techniques: Western Blot, Staining