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

Figure 5. Thermal stress increases cytoplasmic CAR in an HSP70-dependent manner.

Figure 5

Results shown are representative of three independent experiments. (A) HepG2 cells were cotransfected with V5-tagged CCRP (pcDNA3.1/V5-His-mCCRP, 0.3 µg) or empty vector and FLAG-tagged mCAR (pCR3-mCAR-FLAG, 3 µg) or empty vector. 18 hr after transfection, cells were incubated for 1 hr at 42°C or 37°C, followed by treatment at 37°C with DMSO (0.1% DMSO v/v) or MG132 (5 µM in 0.1%DMSO, final concentrations). Cells were then harvested and cytosolic extracts prepared and subjected to immunoblotting analysis with antibodies against the indicated proteins. (B) HepG2 cells were transfected with V5-tagged mouse CAR (pcDNA3.1/V5-His-mCAR, 3 µg) and mouse CCRP (pcDNA3.1/V5-His-mCCRP, 0.3 µg). 18 hr after transfection, cells were incubated for 1 hr at 42°C or 37°C, followed by treatment at 37°C with DMSO for 24 hr. Cell extracts were prepared and then subjected to immunoprecipitation analysis using anti-V5 or anti-IgG (as control) antibodies, followed by immunoblotting analysis using anti-V5 (top panel) and anti-HSP70 (bottom panel) antibodies.