Figure 2. Binding of GAPDH to hTAF_II68 (NTD) in vitro.

(A) Specific association of hTAF_II68 (NTD) with GAPDH. GST-fusion proteins containing the NTDs of hTAF_II68, EWS or TLS were incubated with cell lysates. An aliquot of the inputs (20%) and the pellets from the various pull-downs were analysed on SDS/PAGE (15% gels) and bound GAPDH protein was detected using an anti-GAPDH antibody (MAB374). The identities of the GST-fusion proteins are indicated above the panel. The positions of the molecular mass markers are indicated on the left-hand side. GAPDH is indicated by an arrow on the right-hand side. Three independent experiments were performed, all of which gave similar results. Lane 1, 20% input; lane 2, GST alone; lane 3, GST–hTAF_II68 (NTD); lane 4, GST–EWS (NTD); lane 5, GST–TLS (NTD). IB, immunoblotting; Ab, antibody. (B) Quantitation of the GST-fusion proteins used in the GST pull-down assays. The GST-fusion proteins utilized in the pull-down assays were fractionated by SDS/PAGE (15% gels) and visualized by Coomassie Blue staining. Three independent experiments were performed, all of which gave similar results. Lane 1, GST alone; lane 2, GST–hTAF_II68 (NTD); lane 3, GST–EWS (NTD); lane 4, GST–TLS (NTD).