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. 2014 May 2;9(5):e96092. doi: 10.1371/journal.pone.0096092

Figure 1. Stabilization of CAR in the cytoplasm.

Figure 1

(A) HepG2 cells were transfected with V5-tagged mouse CAR (pcDNA3.1/V5-His-mCAR-V5, 3 µg) and mouse CCRP (pcDNA3.1/V5-His-mCCRP, 0.3 µg), and 18 hours later transfected cells were treated with DMSO (0.1% v/v) or TCPOBOP (250 nM) for 24 hr. Cells were then harvested and cytosolic extracts prepared and subjected to immunoblotting analysis with anti-V5 antibodies. (B) HepG2 cells were transfected with V5-tagged CCRP (pcDNA3.1-mCCRP-V5, 0.3 µg) or empty vector (pcDNA3.1-V5) combined with FLAG-tagged mCAR (pCR3-mCAR-FLAG, 3 µg).18 hours after transfection, cells were treated with DMSO (0.1% v/v), TCPOBOP (250 nM in 0.1% DMSO, final concentrations), MG132 (5 µM in 0.1% DMSO, final concentrations), or TCPOBOP combined with MG132 for 24 hr. Cells were then harvested and cytosolic extracts prepared and subjected to immunoblotting analysis with anti-V5 antibodies (upper panel) and with anti-FLAG antibodies (lower panel). Results shown are representative of three independent experiments.