In vivo homo- and hetero-oligomer formation of human EHD proteins in mammalian cells. (A) HEK 293T cells in 100-mm tissue culture dishes were co-transfected with DNA encoding a single Myc-EHD (2.5 μg) and one EHD-GFP (2.5 μg) construct. Cell lysates were prepared 26–30 h after transfection, rocked overnight at 4°C, and 1 mg aliquots of lysate were subjected to immunoprecipitation (IP) with 3 μg of anti-Myc (9E10) antibody followed by serial anti-Myc and anti-GFP immunoblotting. A negative control (control) using 3 μg of anti-Cbl-b mouse monoclonal IgG1 (lane 9 and 19) was carried out using the lysates transfected (*) as in lane 1 and lane 11, respectively. Further negative controls for the specificity of the co-IP are shown in Additional File 2C. The identity of the lower bands in lanes 15–18 of the anti-Myc blots are unknown. (B) HEK 293T were similarly co-transfected with DNA encoding a single Myc-EHD ΔEH (2.5 μg) and an EHD-GFP (2.5 μg) construct and IPs were carried out as above except that lysates were rocked only 1–2 hours at 4°C following lysis. This exception allowed positive detection of EHD2 ΔEH co-IPs whereas EHD1 ΔEH results were unaffected with further rocking. The identity of the lower bands of the anti-Myc blots in lanes 5–9 and 15–19 are the heavy chain of the mouse IgG (IgH) and are assumed to be masked by and comigrating with Myc-EHD1 ΔEH and Myc-EHD3 ΔEH in lanes 1–4 and 11–14, respectively. All blots are representative of 3 experiments and the lysates used for these IPs are shown in Additional File 2A–2B. Relative molecular weight (MW) markers are indicated in kiloDaltons (kD).