Figure 2
From: Feedback inhibition by RALT controls signal output by the ErbB network
In vitro analysis of RALT interaction with ErbB RTKs. (a) Recombinant GST-RALT cl.52 fusion protein (referred to as GST–RALT) spanning positions 282–396 of RALT was purified onto glutathione–sepharose beads and used in pull-down assays against ErbB receptors solubilized from the indicated NIH 3T3 transfectants. Lysates of parental NIH 3T3 were used as control. Where indicated, cells were treated with 20 ng/ml recombinant NRG-1β for 5 min prior to lysis. Protein complexes were subjected to immunoblot analysis with the indicated antibodies. The bottom portion of the gel was stained with Coomassie blue to control for equal input of GST–RALT. Reference lysates (immunoblotted with anti-ErbB-3 and anti-P-Tyr antibodies) corresponded to 5% of input lysate in GST pull-down experiments. Control experiments (data not shown, see also Figure 3b) indicated that GST alone did not bind to ErbB RTKs. (b and c) NIH-EGFR (b) and NIH-ErbB-4 (c) transfectants were serum starved for 16 h and lysed either without further treatment or after stimulation for 5 min with 20 ng/ml of the indicated ligands. Lysates were subjected to pull-down assays with GST–RALT and immunoblotted with anti-EGFR (b) and anti-ErbB-4 antibodies (c). Reference lysates corresponded to 5% of input lysate in GST pull-down experiments. Input GST–RALT was visualized by Coomassie stain
