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Fig. 3. Erasin localizes to the ER. (A) Immunofluorescence microscopy. (a-c) Indirect immunofluorescence microscopy of HeLa cells probed with the preimmune 130 serum (a) or with the anti-erasin 130-immune serum (b and c). Cells in panels a and b were untransfected whereas those in panel c were transfected with a FL untagged erasin cDNA expression plasmid. (d-f) Colocalization of erasin and calnexin. HeLa cells transfected with C-Myc erasin and revealed with monoclonal anti-Myc antibody (d), endogenous calnexin revealed with rabbit polyclonal anti-calnexin antibody (e), and the result of merging the erasin and calnexin images (f). Bar, 5 µm for all panels. (B) Immunoblots showing distribution of proteins in HeLa cell homogenates fractionated on 0-25% iodixanol gradients. The upper three panels show the distribution of endogenous erasin, calnexin, and golgin-97 proteins. The lower two panels show the distribution of the two different GFP-tagged erasin constructs expressed in HeLa cells. (C) Immunoblots showing erasin is only found associated with a membrane fraction and is not present in soluble, ER-luminal or nuclear fractions. The different fractions were prepared as described in the Materials and Methods. Controls showing fractionation of calnexin, an ER-membrane associated protein, BiP and calreticulin, two ER luminal proteins, and CENP-B, a nuclear protein. (D) HeLa cell lysates were separated into pellet (P) and supernatant (S) fractions in 1x PBS, in 1 M NaCl, in 0.1 M Na2CO3 (pH 11) or in 1% Triton X-100. These fractions were analyzed by immunoblotting using anti-erasin, anti-calnexin and anti-GM130 antibodies. GM130 is a known peripheral Golgi protein used here as a control.
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