FIGURE 9.

Ubiquitination of dopamine receptor types other than the D4 is not mediated by KLHL12. HEK293T cells were transiently transfected with plasmids expressing the HA D4.2 receptor (4 μg), HA D1 receptor (4 μg), HA D2L receptor (4 μg), HA D5 receptor (4 μg), FLAG-tagged ubiquitin (FLAG Ub; 4 μg) and Etag KLHL12 (4 μg), as indicated. 48 h post-transfection, cells were lysed. Small parts of the lysates were subjected to SDS-PAGE and subsequent immunoblotting with the indicated antibodies. The rest of the lysate was subjected to immunoprecipitation (IP) with anti-HA. The washed immunoprecipitates were denatured, and the eluate was subjected to SDS-PAGE. Immunoblotting (IB) with anti-HA confirmed the immunoprecipitation of the various types of HA-tagged dopamine receptors, whereas ubiquitination of these receptors was detected by immunoblotting with anti-FLAG.