K7 induces proteasome-dependent degradation of vGPCR.
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(A) K7 reduces the half-life of vGPCR. ECV cells were transfected with plasmids expressing vGPCR-Flag and K7-V5. Pulse chase, IP, and autoradiography analyses were performed as described in Materials and Methods. The fully glycosylated vGPCR band was quantified and its half-life was calculated. Data (left panel) represent three independent experiments and error bars denote standard deviation. (B) K7-induced vGPCR degradation is dependent on the proteasome. Transfection and pulse chase experiments with ECV cells were performed as described in (A) except cells were harvested at time points as indicated. The numbers at the bottom indicate the relative intensity (top row) of vGPCR band compared to the initial chase time point and standard deviation (bottom row). Data represent three independent experiments. Lac: lactacystin (10 µM); MG: MG132 (20 µM); Ch: chloroquine (50 µM). (C) K7 increases vGPCR ubiquitination. NIH3T3/lenti-puro (Vec) or NIH3T3/lent-vGPCR-Flag (vGPCR) were transfected with plasmids expressing K7-V5 and HA-tagged Ubiquitin (wt), K48R (R48), or K63R (R63). At 36 h after transfection, cells were treated with lactacystin (20 µM) for 6 h. vGPCR was precipitated with anti-Flag sepharose and eluted with Flag peptide for immunoblot with anti-HA (ubiquitin, first panel from left), or eluted with loading buffer for immunoblot with anti-Flag (vGPCR, second panel). WCLs were analyzed by immunoblot with anti-HA (ubiquitin; third panel) and anti-V5 (K7; fourth panel) antibodies. Arrowheads indicated ubiquitinated vGPCR species (second panel).