Ubiquitination of the D4 receptor and KLHL12 in eukaryotic cells.
A, schematic illustration of the D4 receptor-KLHL12-Cul3 E3 ubiquitin
ligase complex. E2 represents the ubiquitin-conjugating enzyme; Roc1 (Rbx1 and
Hrt1) is the RING finger factor; Cul3 serves as the scaffold protein for both
Roc1 and KLHL12. The D4 receptor is targeted to the E3 ligase by the adaptor
protein KLHL12 and functions as a substrate for subsequent ubiquitination by
E2. B, HEK293T cells were transiently transfected with plasmids
encoding the HA D4.2 receptor (4 μg), FLAG-tagged ubiquitin (FLAG
Ub; 4 μg) and Etag KLHL12 (4 μg) as indicated. 48 h
post-transfection, cells were lysed. The lysates were subjected to a first
immunoprecipitation (IP 1) with anti-HA. Bound proteins were eluted
from the washed immunoprecipitates; a quarter of the eluate was subjected to
SDS-PAGE and subsequent immunoblotting (IB) with anti-HA to confirm
immunoprecipitation of the HA D4.2 receptor and anti-FLAG antibody to detect
ubiquitination (top; (FLAG Ub)n-D4.2). Detection
of Etag KLHL12 in the immunoprecipitates was confirmed by immunoblotting with
anti-Etag. The rest of the eluate was diluted with lysis buffer and subjected
to a second immunoprecipitation round (IP 2) with anti-HA. Bound
receptor was eluted from the washed immunoprecipitates. The eluate was again
subjected to SDS-PAGE and subsequent immunoblotting with anti-HA to confirm
the second immunoprecipitation of D4.2 receptor and anti-FLAG to detect
specific ubiquitination of the D4.2 receptor (bottom). The exclusion
of KLHL12 from the second immunoprecipitates was confirmed by immunoblotting
with anti-Etag. *, the signal is a combination of IgG, a nonspecific signal
from the anti-HA antibody, and a D4-specific signal in lanes 1,
3, 4, and 5. C, HEK293T cells were
transiently transfected with plasmids expressing Etag KLHL12 (4 μg), FLAG
Ub (4 μg), and HA Cul3 (4 μg). 48 h post-transfection, cells were lysed.
A small fraction of each lysate was subjected to SDS-PAGE to confirm the
expression of FLAG Ub and HA Cul3 by immunodetection with anti-FLAG and
anti-HA, respectively. The rest of the lysates were subjected to a first
immunoprecipitation using anti-Etag. Bound proteins were eluted from the
beads; 20% of the eluate was subjected to SDS-PAGE and subsequent
immunoblotting with anti-Etag to confirm immunoprecipitation of Etag KLHL12
and with anti-FLAG to detect ubiquitinated Etag KLHL12 (top;
(FLAG Ub)n-KLHL12). The rest of the eluate was diluted
with lysis buffer and subjected to a second immunoprecipitation round with
anti-Etag. Bound Etag KLHL12 was eluted from the washed immunoprecipitates and
again subjected to SDS-PAGE. The second immunoprecipitation of Etag KLHL12 was
confirmed by subsequent immunoblotting with anti-Etag, whereas specific
ubiquitination of Etag KLHL12 was detected by immunoblotting with
anti-FLAG.