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. 2012 Dec 4;12(2):499–514. doi: 10.1074/mcp.M112.020768

Fig. 2.

Fig. 2.

Identified proteins REPS1 and BCR interact with the carboxyl region of Numb: A, Schematic diagram showing GST fusion proteins used for in vitro pull down assays in panels C and E. B, HEK293T cells were co-transfected with MYC-BCR and HA-Numb isoforms as indicated. Cell lysates were immunoprecipitated with antibodies directed against the HA epitope tag of Numb and protein complexes were separated by SDS-PAGE and analyzed by immunoblot. C, GST fusion proteins of the Numb PTB domain (GST-Nb PTB), the carboxyl terminal half of Numb including (GST-Nb 184–603) or excluding (GST-Nb 184–555) the exon9 region, or GST alone were purified and incubated with cell lysate from HEK293T cells overexpressing MYC-BCR. Complexes were separated by SDS-PAGE and analyzed by immunoblot using anti-MYC antibody. D, HEK293T cells were cotransfected with HA-REPS1 and either FLAG-Numb isoforms or empty vector controls as indicated. Lysates were incubated with antibodies directed against HA epitope tag of REPS1 or FLAG epitope. Protein complexes were eluted, separated by SDS-PAGE, and analyzed by immunoblot using antibodies for FLAG (Numb). E, GST fusion proteins GST-Nb PTB, GST-Nb 184–603 or GST alone were purified and incubated with cell lysate from HEK293T cells overexpressing HA-REPS1. Complexes were separated by SDS-PAGE and analyzed by immunoblot using an anti-HA antibody.