Eps15 homology domain-containing (EHD) 1–4 proteins show differential localization in skeletal muscle. The tibialis anterior (TA) skeletal muscles of three wild-type (WT) and three EHD1−/− mice were teased into small bundles for immunofluorescence and visualization via confocal microscopy. α-bungarotoxin (BTX) labeling of AChRs denote neuromuscular junctions (NMJs) in all images. EHD2, 3, and 4 are expressed in WT and EHD1−/− mice. Panel (A) EHD2 localized to skeletal muscle vasculature endothelium. (B) EHD3 localized to synaptic regions adjacent to theBTX labeling and in the vasculature, but to a lesser extent than EHD2. (C) EHD4 localized to the synaptic cleft with BTX in addition to adjacent extrasynaptic regions and perinuclear regions. (D) EHD 1–4 expression in skeletal muscle lysates was analyzed by western blotting. ( E ) EHD1-4 antibodies also recognized proteins of similar mass in Torpedo californica electric organ lysates. Blue, DAPI; green, BTX Alexa Fluor 488 conjugate; red, anti-rabbit Alexa Fluor 555 conjugate.Scale bars: 20 μm. A larger reference bar was added above the scale bars.