FIGURE 3.

GPNMB preferentially binds to mutated EGFR and facilitates its downstream signaling without ligand stimulation. A, Various EGFR were co‐transfected with or without GPNMB into H1299 cells, and the downstream signaling of EGFR was examined by immunoblotting. “Mock” represents cells without any manipulation, “–” indicates vector control. B, FLAG‐tagged EGFR mutants and myc/His‐tagged GPNMB were co‐transfected into H1299 and immunoprecipitated (IP) with anti‐FLAG antibodies. The precipitated amounts of EGFR and GPNMB were examined by immunoblotting (arrowheads: the precipitated product). C, Total lysates from NSCLC cells with different EGFR status were used to perform immunoprecipitation with anti‐GPNMB or IgG antibodies and the bindings between EGFR and GPNMB were examined by immunoblotting. Left: Input proteins. Right: The precipitated amounts of various EGFR and GPNMB (arrowheads) were determined and quantification shown under the blot. D, Domain mapping of GPNMB with wild‐type and mutated EGFR. Top: FLAG‐tagged GPNMB deletion mutants, the interactions of each fragment are shown on the right. Bottom: The resulting blots of domain mapping (*, indicates the band of each truncated GPNMB)