Subcellular distribution of U2AF65. (a) RT-PCR analysis of spliced and unspliced β-actin mRNA in cytoplasmic (Cyt) and nuclear (Nuc) fractions isolated from HeLa cells. Primer sequences on actin RNA and size of expected amplification products are depicted below the gel. (b) Western blot analysis of the cytoplasmic (Cyt) and nuclear (Nuc) fractions using antibodies against the indicated proteins. Molecular weight markers are shown on the left. Coomassie staining of the gel used for blotting confirms that both fractions contain similar amounts of total protein. (c) Agarose gel electrophoresis of total RNA extracted after Nycodenz gradient fractionation of cytoplasmic samples. rRNA bands are indicated on the right. Numbers indicate gradient fractions from low to high density. (d) Semiquantitative RT-PCR analysis of actin mRNA in the gradient fractions. (e) Western blot analysis of gradient fractions using anti-U2AF65 and anti-PTB antibodies. Molecular weight markers are indicated on the left. The 65 kDa band in the membrane after reprobing for PTB corresponds to residual signal from U2AF65 detection. (f) Western blot analysis of gradient fractions and input samples using anti-U2AF65 antibody. Extracts were either mock-treated (control) or incubated with RNase A before fractionation. Molecular weight markers are indicated on the left. Arrow points to the intermediate density fraction where U2AF65-containing complexes accumulate. PTB, polypyrimidine tract binding protein; RT-PCR, reverse transcription polymerase chain reaction; U2AF, U2 small nuclear RNP auxiliary factor.