Figure 5

EHD protein expression and EHD1 localization during mouse testis development. (A, upper panel) Aliquots of 50 μg testis lysates from post-natal day 10-42 mice were separated using 8% SDS-PAGE and a Western blot was performed using affinity purified antibodies raised against EHD1 (described in Methods), followed by serial reprobing with antisera raised against EHD proteins as described previously [7]. β-Actin served as a loading control. The * denotes bands that bled through from the previous blot. (A, lower panel) Aliquots of 20 μg immortalized mouse TM4 Sertoli cell and mouse embryonic fibroblast (MEF, Ehd1^{fl-Neo/fl-Neo}) lysates were treated similarly except the membrane was probed with antisera that recognize EHD1 and EHD4 followed by EHD2. (B, C) Immunohistochemistry was carried out to determine EHD1 localization in day 10 and day 30 formalin-fixed testis sections from WT and Ehd1^-/- mice. EHD1 expression can be seen as brown staining; nuclei are counter-stained with hematoxylin (blue). Panels A and B contained affinity purified anti-EHD1 primary antibodies while panels C and D lacked primary antibodies (control). Insets in panel A are enlarged micrographs of the highlighted cells. Note: similar seminiferous tubules from adjacent sections can be seen in A and C as well as B and D; denoted by asterisks (**). Sc - Sertoli cell, Sg - spermatogonia. Scale bar = 100 μm.