Eps15 homology domain-containing (EHD)4 expression is increased in EHD1−/−muscle. The tibialis anterior (TA) skeletal muscle of three wild-type (WT) and three EHD1−/− mice were teased into small bundles for immunofluorescence labeling with anti-EHD4 antibody and z-stacks were acquired to assess signal throughout the entire myofiber diameter/thickness via confocal microscopy. α-bungarotoxin (BTX) labeling of AChRs denote NMJs. EHD4 signal intensities were compared in myofibers (n = 5) and at neuromuscular junctions (NMJs) (n = 10) from WT and EHD1−/− mice (n = 3 per group) using identical acquisition parameters. (A) The z-section with the highest signal for EHD4 (red channel). (B) Intensity projections of the EHD4 signal from representative NMJs in (A). 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. (C) The average intensity of EHD4 expressed in WT and EHD1−/− mice. The bars represent the average vector measurements of the intensity with standard error from myofibers and NMJs in each group. Statistical significance was measured by Student’s t-test (P < 0.05).