Figure 4
From: Feedback inhibition by RALT controls signal output by the ErbB network
In vivo analysis of physical interaction between RALT and the ErbB-2.ErbB-3 NRG-1 receptor. (a) ErbB-2 and ErbB-3 were transiently expressed in 293 cells either alone or in combination. Where indicated, either RALT or RALT ΔEBR were coexpressed with ErbB RTKs. After 2 h serum starvation, cells were stimulated at 37°C with either carrier solution or 20 ng/ml NRG-1β before lysis. Equal amounts of each lysate were analysed by immunoblot with anti-ErbB-2 and anti-ErbB-3 antibodies to control for receptor expression. Lysates were subjected to immunoprecipitation with anti-RALT 19C5/4 monoclonal antibody followed by blot analysis with anti-RALT and anti-ErbB-3 antibodies. Filters were subsequently reprobed with anti-ErbB-2 antibodies. (b) The ΔEBR mutation does not affect the ability of RALT to interact with Grb-2. HA-tagged GRB-2 was expressed in 293 cells either alone or in combination with RALT or RALT ΔEBR. Equal amounts of each lysate and anti-RALT immunoprecipitates were immunoblotted with anti-HA tag 12CA/5 and anti-RALT 19C5/4 monoclonal antibodies
