Eps15 homology domain-containing (EHD)1 is expressed in mouse skeletal muscle and localizes to the primary synaptic clefts of neuromuscular junctions. (A) Western blot of two WT and two EHD1−/− skeletal muscle lysates probed with peptide-specific antibodies against EHD1 and EHD4 showedexpression in skeletal muscle and verified the loss of EHD1 in null mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (B) 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 NMJs in all images. EHD1 localized to the NMJ and delineated the primary synaptic cleft similar to BTX labeling. (C) Because the EHD1 antibody crossreacts with EHD4, imparting low background signal, EHD1−/− fibers were labeled with anti-EHD1 to show the specificity of the localization. Blue, DAPI; green,BTXAlexa Fluor 488 conjugate; red, anti-green fluorescent protein Alexa Fluor 555 conjugate. Scale bars: 20 μm. A larger reference bar was added above the scale bars.