Marcu A , Bassit B , Perez R , Piñol-Roma S .

GM53468 NIGMS NIH HHS

	Fig. 1. Affinity chromatography of Xenopus laevis proteins on ssDNA-cellulose. Whole X. laevis A6 cell lysate proteins were fractionated on a ssDNA-cellulose column as described in the text. Proteins in the various fractions were resolved by SDS-PAGE and visualized by staining with silver. Lanes are as follows: Total, starting material; F.T., unbound (flow-through) fraction; Heparin, proteins in a wash with 1mg/ml heparin; 0.2 M and 2.0 M NaCl, proteins eluted with the indicated NaCl concentrations. Positions of molecular mass standards are shown on the left.
	Fig. 2. Immunoblot analysis of X. laevis and human proteins with anti- Xenopus ssDNA-binding protein monoclonal antibodies. Whole X. laevis lysate (lanes Xep.) and whole human HeLa cell lysate (lanes Human) proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with the indicated monoclonal antibodies. Positions of molecular mass standards are indicated on the left.
	Fig. 3. Immunofluorescence microscopy staining of X. laevis cells with monoclonal antibodies to ssDNA-binding proteins. X. laevis kidney epithelial (A6) cells grown on glass slides were fixed, permeabilized, and immunostained with the indicated monoclonal antibodies. Representative fields of cells are shown in each case, together with the corresponding phase microscopy image. Bar, 10 mm.
	Fig. 4. Two-dimensional gel electrophoresis and immunoblot analysis of X. laevis ssDNA-binding proteins. Proteins from the 2.0 M NaCl eluate shown in Fig. 1 were resolved by two-dimensional gel electrophoresis, using NEPHGE in the first dimension and SDS-PAGE in the second dimension. The separated proteins were visualized by silver staining (left panel), by immunoblotting with 10D1 (middle panel), or by immunoblotting with 1A5 (right panel). Arrowheads in the left panel point to proteins reactive with the 10D1 monoclonal antibody. Arrows in the left and right panels point to the position of the 1A5-reactive proteins. Positions of molecular mass standards are indicated on the left.
	Fig. 5. Identification of human hnRNP proteins as antigens for 10D1 and 1A5. HeLa cell nuclear extract was fractionated by ssDNA-cellulose chromatography, and proteins eluted with 2.0 M NaCl were resolved by two-dimensional gel electrophoresis. Proteins were visualized by silver staining (left panel), or by immunoblotting with 10D1 (middle panel) or 1A5 (right panel). The positions of hnRNP A1, A2, B1, B2, and K, are indicated on the silver-stained gel.
	Fig. 6. Immunoblot analysis of proteins crosslinked to poly(A)+ RNA in vivo. Living X. laevis A6 cells were exposed to ultraviolet light, and poly(A)+ RNA was then isolated with its covalently bound proteins. Proteins were released from the crosslinked complexes with RNase digestion, resolved by SDS-PAGE, and probed with the indicated antibodies. Lanes are labeled as follows: Total: whole A6 cell proteins without crosslinking and oligo(dT) selection; +UV: oligo(dT)-selected complexes from cells exposed to UV light; -UV: control experiment carried out as shown in the +UV lanes except that exposure of the cells to UV light was omitted.
	Fig. 7. Binding of 10D1 to X. laevis hnRNP A2 transcribed and translated in vitro. X. laevis hnRNP A2 was transcribed from the corresponding cDNA and translated in vitro in a rabbit reticulocyte lysate in the presence of 35Smethionine. The translated protein was subsequently immunoprecipitated using either 10D1 or the anti-hnRNP A1 4B10 monoclonal antibody, as indicated. Total translation reaction equivalent to one-fifth of the material used for immunoprecipitation was resolved on the same gel for comparison (lane �A2 cDNA�). The translation products were visualized by autoradiography. Lane �No DNA�: total translation product from an identical reaction performed in parallel in which no exogenous DNA was added.
	Fig. 8. Immunopurification of X. laevis hnRNP complexes from somatic cells. Immunopurifications with the 10D1 antibody were carried out from X. laevis cell lysate prepared in isotonic buffer containing 0.5% Triton X-100 (panel TNX-100) or in the presence of the ionic detergent Empigen BB (panel Empigen). Immunopurified complexes were resolved by two-dimensional gel electrophoresis, and proteins were visualized by silver staining. The positions of known hnRNP proteins (determined by immunoblot analyses not shown) are indicated. Proteins that also bind ssDNA are indicated with arrows. Ig h.c. and Ig l.c. refer to heavy and light chains, respectively, of the 10D1 antibody. Asterisks indicate proteins originating also from the antibody preparation. Positions of molecular mass standards are indicated on the left.
	Fig. 9. Immunopurification with 10D1 from Xenopus oocyte nuclear and cytoplasmic fractions. X. laevis oocytes (ca. 20 oocytes) were manually dissected into nuclear and cytoplasmic fractions, and immunopurifications were carried out from each fraction with the 10D1 antibody as described in Fig. 8. Proteins in the immunopurified complexes were resolved by two-dimensional gel electrophoresis and visualized by silver staining also as in Fig. 8. Left panel: complexes immunopurified from the nuclear fraction. The positions of hnRNP A2/B1/B2 and hnRNP K are indicated, as are the positions of the heavy and light chains of the antibody used for immunopurification. Arrows point to additional proteins in the complex that co-migrate with those observed in complexes immunopurified from A6 cells (compare with Fig. 8). Right panel: immunopurification from the cytoplasmic fraction of an identical number of oocytes as in the left panel.