Figure 2.

Evidence that A1 is part of the nuclear protein complexes formed by 219T probe. (A) EMSA analysis of the formation of complex a by the fractions eluted from the 219T affinity column. The column fractions were numbered by E1 to E8. (B) Identification of A1 in the 219T column eluates. Fractions were analyzed by SDS–PAGE followed by Coomassie staining; the indicated band was in gel digested and the resulting peptides analyzed by mass spectrometry. Bands identified as hnRNPs A2/B1 and A1 are indicated. (C) Evidence that A1 is specifically retained by 219T column. Fractions eluted from the 219T column and the other two control columns (NR1 and NR2) were assayed by western blot analysis using an anti-A1 antibody. The arrow indicates the position of A1. (D, E and F) Evidence that A1 forms part of the main nuclear complex formed by 219T in the presence of nuclear cell extracts. In (D), fraction E8 eluted from the 219T affinity column was incubated with 219T and 219G probes, subjected to UV-induced crosslinking and analyzed by SDS–PAGE followed by autoradiography (left panel). Nuclear extracts from Jurkat cells were incubated with 219T probe and subjected to immunoprecipitation using an anti-A1 (α-A1) or a control antibody (α-APP); the precipitated material was then subjected to UV-induced crosslinking, followed by SDS–PAGE analysis and autoradiography. The positions of the A1-containing, specific 219T-protein complex (p50) and of the non-specific complex (p52) are indicated. In (E), Jurkat nuclear extracts inmunodepleted with α-A1 and α-APP antibodies were incubated with 219T and 219G probes, subjected to UV- induced crosslinking, and analyzed by SDS–PAGE and autoradiography. In (F), Jurkat nuclear extracts (N.E.) inmunodepleted with α-A1 and α-APP antibodies were incubated with 219T probe and subjected to EMSAs. The position of complex a is indicated with an arrow. Molecular weight markers are indicated on the left in (B), (C) and (D). A non-specific band is indicated by an asterisk in (D) and (E).