KLHL12 interacts with the D4 receptor in eukaryotic cells. HEK293T
cells were transiently transfected with plasmids expressing HA D4.2 receptor
(5.5 μg) and/or Etag KLHL12 (5.5 μg), as indicated. 48 h
post-transfection, cells were lysed. 5% of the lysates were subjected to
SDS-PAGE and subsequent immunoblotting (IB) with anti-HA and
anti-Etag to detect expression of HA D4.2 receptor and Etag KLHL12,
respectively (bottom). The rest of the lysates were subjected to
immunoprecipitation (IP) with anti-HA. The D4-KLHL12 association was
examined through subsequent immunoblotting with anti-Etag. Immunoprecipitation
of the HA D4.2 was confirmed by subsequent immunoblotting with anti-HA. *,
this signal represents a combination of immunoglobulin G, a nonspecific
binding signal from the anti-HA antibody, and a D4-specific signal in
lanes 2 and 4. **, a combined nonspecific signal from
anti-HA and a D4-specific signal in lanes 2 and 4.