Identification of hnRNP L as the major protein in nuclear extracts of B16F10 cells that binds CA6 or CA21 mRNA probes. (A) A ³²P-labeled mRNA probe representing either the FVB/NJ sequence (CA6) (lane 1, 1 μg; lane 2, 5 μg) or the BALB/cJ sequence (CA21) (lane 3, 1 μg; lane 4, 5 μg) was mixed with a nuclear extract from B16F10 cells and subjected to UV irradiation. The mRNA-protein complexes were then separated by SDS-PAGE, and proteins that had bound each probe were visualized by autoradiography. The major complex has a MW_App of 64 kDa, the mobility expected for hnRNP L. On the basis of optical scanning and densitometry, the intensity of the analogous band obtained with the FVB/NJ CA6 probe is approximately 50% of that obtained with the BALB/cJ CA21 probe. (B) Immunoprecipitation of mRNA-protein complexes with anti-hnRNP L. A ³²P-labeled CA6 mRNA probe (lane 1, 1 μg; lane 2, 5 μg) or CA21 mRNA probe (lane 3, 1 μg; lane 4, 5 μg) was mixed with a nuclear extract from B16F10 cells and subjected to UV irradiation. mRNA-protein complexes were then precipitated with anti-hnRNP L. The immunoprecipitate were isolated and washed, and the components of the precipitate were solubilized in SDS and separated by SDS-PAGE. Proteins that had bound probe were visualized by autoradiography. Once again a single major band with a MW_App of 64 kDa is clearly visible in lane 4. As a negative control, the procedure was repeated with anti-Sp3 (lane 5). In this case, no labeled protein bands were detected in the immunoprecipitate. The results depicted were derived from 1 experiment that is representative of 3 independent experiments.