![Figure 1. Autophagy machinery is present in murine and human platelets. (A) Western blot analyses of the autophagy machinery in freshly prepared mouse and human platelets and in banked human platelets. Extracts from mouse and human platelets were loaded in each lane and probed with antibodies to the indicated proteins. Atg5+/+ and Atg5−/− mouse embryonic fibroblasts (MEFs) were used as additional molecular weight markers for LC3I (unconjugated form) and LC3II (phosphatidylethanolamine-conjugated form), as Atg5−/− MEFs produced LC3I but not the faster-migrating LC3II. Atg5−/− MEFs also had higher p62 levels than Atg5+/+ MEFs because of impaired autophagy. Note that the loading amounts for the MEFs were much lower than those for platelets to have comparable LC3II levels. As a result, and as platelets have high actin content than the MEFs, both actin and p62 levels in the MEF samples are much lower than in platelets. The images shown are representative of at least 3 independent experiments. Full images of all blots are shown in supplemental Figure 1. (B) Platelets from the autophagy reporter mice (ie, GFP-LC3/+, Becn1-EGFP/+, EGFP-Atg5/+) but not wild-type mice show GFP-LC3-positive, EGFP-Atg5-positive, and Beclin 1-EGFP-positive puncta, respectively (arrows), which are autophagosome-related structures including isolation membranes, autophagosomes, and autolysosomes. Samples were visualized for GFP or EGFP fluorescence (fluorescein isothiocyanate [FITC] channel) and differential interference (DIC). The images shown are representative of at least 2 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/126/10/10.1182_blood-2014-09-598722/4/1224f1.jpeg?Expires=1725046245&Signature=mWfnWAcuqkwCBLTgkf3~dH292ixX0tStvT27GGDeb1ZJ31WIjLl6w6ZvRKaxwYO5Z-AsqqmsC81pad7UU3E0CGwo8op5SnJBRvOXfRyWjaWaUlAu32j2ogXwqdBIIv35JD5umIa1fpwmajXvoBPJxXgA7QsGPD~EO8PM-i0y~~hjKY3XKtYsZl9tJHK7O6VnLXrVWuXPV8dxyDHYON3EtXaWCg0HG4234pO46gxmK7yXmRmVvMvWjvaMI9hR7oY0ZxDFF--OOtz8jwkyIUQclj9u5cVnV35nLPrcCPtRKlse9Bm4WV2a0gUAppeK5yBXMD6bVfN0qomKXy2XXlmo8Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Autophagy machinery is present in murine and human platelets. (A) Western blot analyses of the autophagy machinery in freshly prepared mouse and human platelets and in banked human platelets. Extracts from mouse and human platelets were loaded in each lane and probed with antibodies to the indicated proteins. Atg5+/+ and Atg5−/− mouse embryonic fibroblasts (MEFs) were used as additional molecular weight markers for LC3I (unconjugated form) and LC3II (phosphatidylethanolamine-conjugated form), as Atg5−/− MEFs produced LC3I but not the faster-migrating LC3II. Atg5−/− MEFs also had higher p62 levels than Atg5+/+ MEFs because of impaired autophagy. Note that the loading amounts for the MEFs were much lower than those for platelets to have comparable LC3II levels. As a result, and as platelets have high actin content than the MEFs, both actin and p62 levels in the MEF samples are much lower than in platelets. The images shown are representative of at least 3 independent experiments. Full images of all blots are shown in supplemental Figure 1. (B) Platelets from the autophagy reporter mice (ie, GFP-LC3/+, Becn1-EGFP/+, EGFP-Atg5/+) but not wild-type mice show GFP-LC3-positive, EGFP-Atg5-positive, and Beclin 1-EGFP-positive puncta, respectively (arrows), which are autophagosome-related structures including isolation membranes, autophagosomes, and autolysosomes. Samples were visualized for GFP or EGFP fluorescence (fluorescein isothiocyanate [FITC] channel) and differential interference (DIC). The images shown are representative of at least 2 independent experiments.
