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Figure 6


Fig. 6. Specificity of N-terminal control of TIP47 binding to CLDs. (A) Specificity of TIP47 antibodies. Extracts of cultures stably expressing the TIP47-ADPH-VSV or ADPH-TIP47-VSV chimeric protein were immunoblotted with chicken anti-human TIP47 antibody ({alpha}hTIP47), rabbit antibodies to the N-terminus of mouse TIP47 ({alpha} N-term mTIP47) or mouse anti-VSV antibody ({alpha}VSV). {alpha}hTIP47 detected a single species at ~47 kDa in cultures of both chimeric cell lines (calculated molecular weight of hTIP47=47032.88; ProSite, http://ca.expasy.org/tools/dna.html). {alpha} N-term mTIP47 and {alpha}VSV antibodies detected a single species of ~52 kDa in TIP-ADPH-VSV cultures (calculated molecular weight=50907.48) and a single species of ~49 kDa (calculated molecular weight=48019.52) in ADPH-TIP47-VSV cultures. (B) Immunolocalization of endogenous TIP47 (green, a-d) and chimeric proteins in ADPH-TIP-VSV (red, a,b) and TIP-ADPH-VSV (red, c,d) in cells after a 20-hour incubation in the absence (Cont, a,c) or presence of 300 µM OA (b,d). TIP47 was localized by staining with {alpha}hTIP47; chimeric proteins were localized by staining with mouse anti-VSV; nuclei were stained with DAPI (blue). All experiments were performed at least three times, with similar results. Multiple images were captured in each experiment; representative images are shown. Images were originally taken at 600x magnification. Scale bar: 20 µm.





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