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.