Sam68 and hnRNP L proteins associate via protein-protein interactions and are not bridged by nucleic acids. (A) LNCaP cell nuclear extracts were subjected to IP using either anti-Sam68 antisera or an anti-hnRNP L monoclonal antibody. Recovered material was subjected to Western analysis with antibodies specific to hnRNP L (left panel) or Sam68 (right panel). (B) HEK293 cells were transfected with expression vectors for FLAG-Sam68 or FLAG alone, and hnRNP L-GST, and subjected to IP using anti-FLAG M2 agarose. Recovered material was subjected to Western analysis with antisera to GST. (C) HEK293 cell nuclear extracts were fractionated on sucrose gradients before (upper panel) or after (lower panel) treatment with MNase to digest nucleic acids. Fraction 20 (containing low molecular weight material) was taken from the top of the gradient, and fraction 1 (containing high molecular weight material) was taken from the bottom. Pelleted material is indicated as P. The migration of individual proteins in each fraction was monitored by SDS-PAGE and Western analysis. The mobility of size markers on the gradients is shown.