Figure 3

Subcellular localization of the mammalian COP1 (A) Detection of endogenous HsCOP1 protein by immunoprecipitation and immunoblot. Whole cell lysates of HeLa cells were immunoprecipitated with purified anti-COP1 antibodies from rabbit 1 (R1) or preimmune serum (Pre) as indicated, separated by SDS-PAGE, and then immunoblotted with anti-COP1 antibodies from rabbit 1, 2, and 3. The arrowheads mark the endogenous HsCOP1 protein and IgG. R1, blot probed with purified COP1 antibodies from rabbit 1; R2, blot probed purified COP1 antibodies from rabbit 2; R3, blot probed with purified COP1 antibodies from rabbit 3; Pre, preimmune serum was used for IP.(B) Localization study of endogenous HsCOP1 by subcellular fractionation. Subcellular fractions of HeLa cells were either immunoprecipitated with anti-COP1 antibodies first, or were directly separated (for all other control proteins) by SDS-PAGE and immunoblotted with antibodies against COP1, Hsp110, Hsp90, Lamin A/C, nucleoporins (mAb414), CSN2 and CSN6 from the top to the bottom panels as indicated. H, homogenate; C, cytosol; MI, mitochondria; MS, microsomes; N, nuclei; NP, nucleoplasm; NE, nuclear envelopes. (C) GFP fluorescence analysis of the subcellular localization of GFP-tagged MmCOP1 protein. N-terminal GFP-tagged MmCOP1 expression construct was transfected into COS7 cells. The cells were fixed with 4% paraformaldehyde 24 hours after transfection. Type I to III are representative of three distinct types of localization patterns, while +Triton indicates cell treated with 1% Triton in PBS for 10 min on ice and then washed three times with PBS before fixation. The subcellular localization of GFP-COP1 was observed under Zeiss Axiophot fluorescence microscope (top). The nuclei were stained by 1% DAPI in anti-fade reagent (middle). The merged pictures of the DAPI and GFP fluorescence images are shown at bottom. The same magnification was used for all the images and the scale bar for 10 micrometer was indicated within the top left image.