Figure 3
Figure 3. Platelet activation leads to LC3II reduction. (A) Western blot analysis of LC3II in the murine and human platelets before and after stimulation (0.1 U/mL thrombin, 30 minutes), with various anti-LC3 antibodies. Cell extracts from Atg5+/+ and Atg5−/− MEFs were used as additional molecular weight markers to indicate the migration positions of LC3I and LC3II. Note that the loading volumes for the MEFs were optimized to have comparable LC3II levels, and platelets have high actin content. Thus, both actin and p62 levels in the MEF samples were below detection limits. The images shown are representative of at least 2 independent experiments. (B) Western blot analyses of LC3II in human platelets before and after stimulation with various agonists. Platelets were stimulated for 30 minutes with thrombin (0.1 U/mL), CVX (0.1 µg/mL), PAR1 or PAR4 peptide (100 µM), ADPβS (10 µM), collagen (1 µg/mL), A23187 (10 µM in DMSO), and U46619 (20 µM in DMSO). N = 2∼8 for various inhibitors. (C) Western blot analysis of LC3II, before and after stimulation, in human platelets pretreated with inhibitors of platelet activation. Platelets were pretreated for 60 minutes with U-73122 (20 µM), U-73343 (inactive analog of U-73122, 20 µM), BAPTA-AM (100 µM), Ro-31-8220 (10 µM), or PP2 (100 µM) and then stimulated for 30 minutes with either thrombin (0.1 U/ml) or CVX (0.1 µg/mL). Of note, U-73343, Ro-31-8220, and PP2 increased LC3II levels in resting platelets for an unknown reason. All inhibitor stock solutions were prepared in DMSO. N = 3 for all inhibitors. For B and C, *P < .05 by the Student t test.

Platelet activation leads to LC3II reduction. (A) Western blot analysis of LC3II in the murine and human platelets before and after stimulation (0.1 U/mL thrombin, 30 minutes), with various anti-LC3 antibodies. Cell extracts from Atg5+/+ and Atg5−/− MEFs were used as additional molecular weight markers to indicate the migration positions of LC3I and LC3II. Note that the loading volumes for the MEFs were optimized to have comparable LC3II levels, and platelets have high actin content. Thus, both actin and p62 levels in the MEF samples were below detection limits. The images shown are representative of at least 2 independent experiments. (B) Western blot analyses of LC3II in human platelets before and after stimulation with various agonists. Platelets were stimulated for 30 minutes with thrombin (0.1 U/mL), CVX (0.1 µg/mL), PAR1 or PAR4 peptide (100 µM), ADPβS (10 µM), collagen (1 µg/mL), A23187 (10 µM in DMSO), and U46619 (20 µM in DMSO). N = 2∼8 for various inhibitors. (C) Western blot analysis of LC3II, before and after stimulation, in human platelets pretreated with inhibitors of platelet activation. Platelets were pretreated for 60 minutes with U-73122 (20 µM), U-73343 (inactive analog of U-73122, 20 µM), BAPTA-AM (100 µM), Ro-31-8220 (10 µM), or PP2 (100 µM) and then stimulated for 30 minutes with either thrombin (0.1 U/ml) or CVX (0.1 µg/mL). Of note, U-73343, Ro-31-8220, and PP2 increased LC3II levels in resting platelets for an unknown reason. All inhibitor stock solutions were prepared in DMSO. N = 3 for all inhibitors. For B and C, *P < .05 by the Student t test.

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