Figure 2. Role of Calpain in ApoE-Induced ABCA1 Protein.

(A) Macrophages were treated with 100 μg/ml cycloheximide (CHX) or vehicle medium in the presence or absence of 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h incubation with 10 μg/ml apoE3 or medium alone. (B) Macrophages were treated with vehicle medium (Veh), 50 μM ALLN, 1 mM leupeptin (Lpeptn), 10 μM aprotinin (Aprotn), 20μM pepstatin (Peptn), or 5 μM lactacystin (Lactyn) for 2 h. (C) Macrophages were treated with 50 μM ALLN or vehicle medium in the presence or absence of 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h incubation with 10 μg/ml apoE3 or control medium. (D) Macrophages were treated with or without 4 μM Akt inhibitor X (Akt X) for 2 h, followed by 2 h treatment with 10 μg/ml apoE3 or control medium alone. The cells were then treated with 3 μg/ml of calpain-1 or vehicle medium. ABCA1 (A1) protein was detected by immunoblotting, and quantified relative to GAPDH (A–C) or CD-MPR (D). (E) Macrophages were treated with 4 μM Akt X or vehicle medium for 2 h, followed by 2 h treatment with 10 μg/ml apoE3 or control medium. Calpain activity was measured with a calpain activity kit. Data represent mean ± SE of 3–4 independent experiments. (A) * P< 0.05 vs. cells without apoE3 treatment in the vehicle or CHX treatment group, respectively; † P< 0.05 vs. cells treated with apoE3 but without Akt X in the vehicle or CHX group, respectively; and ‡P< 0.05 vs. similarly treated cells without CHX. (C) * P< 0.05 vs. cells treated with medium alone; and †P< 0.05 vs. cells treated with apoE3 alone. (D) * P< 0.05 vs. cells without apoE3 treatment in the vehicle or calpain treatment group; ^†P< 0.05 vs. cells treated with apoE3 but without Akt X in the vehicle or calpain group, respectively; and ^‡ P< 0.05 vs. similarly treated cells without calpain.