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Fig. 1. ProAR translocates from the plasma membrane to the inner nuclear membrane. (A) Schematic representation of human AREG gene products. The AR coding region is translated as a precursor form (pre-proAR) with five structural domains consisting of predicted 252 amino acids (Plowman et al., 1990). The signal sequence is trimmed off, and the resulting protein is expressed on the plasma membrane as proAR whose N-terminal sequence is not yet determined (*). In response to various stimuli, proAR is shed at the juxtamembrane domain, resulting in the production of AR and AR-CTF. Anti-AR-N and anti-AR-C pAbs specifically recognize the extracellular and the cytoplasmic domains of proAR, respectively. The single line is the epitope region of anti-AR-N pAb, and double line is that of anti-AR-C pAb. (B) HeLa cells were transfected with a full-length AREG plasmid, then incubated with or without TPA. Cells were immunostained with anti-AR-N or anti-AR-C pAb. (C) HeLa cells transiently expressing AREG were incubated with 20 µg/ml cycloheximide for 60 minutes. The cells were then treated with TPA and immunostained with anti-AR-N pAb. (D) HeLa cells transiently expressing AREG were permeabilized with digitonin and then fixed. The cells were re-permeabilized with (left panels) or without (right panels) Triton X-100, and immunostained with anti-AR-C (green) or/and anti-lamin B (red) pAbs. The lower panels are high magnification images of the boxed regions labelled 1 and 2. Scale bars: 5 µm. (E) Ultra-thin sections were stained with anti-V5 mAb and 15 nm gold-conjugated secondary antibodies. Right panel, without TPA; middle panel, with TPA; left panel, high magnification image of boxed region in middle panel. PM, plasma membrane; NE, nuclear envelope. Scale bars: 200 nm.
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