FIGURE 2.

EGFR localizes to the mitochondria. A, 10T1/2 cells co-overexpressing EGFR and c-Src were cultured in serum-free media for 24 h, left untreated or stimulated with 100 ng/ml EGF for 20 min, and processed for immunoelectron microscopy analysis as described under “Experimental Procedures.” Arrows indicate positive EGFR staining. B, mitochondria were purified from rat brain and subjected to immunoelectron analysis as described under “Experimental Procedures.” Samples were stained with either anti-EGFR antibody or no primary antibody as a control. Arrows indicate positive EGFR staining in the mitochondria. C, lysates from EGF-stimulated or non-stimulated 10T1/2 cells co-overexpressing EGFR and c-Src were prepared as described under “Experimental Procedures.” Two mg of protein lysate was immunoprecipitated (IP) with EGFR, Tom40 (goat), Tom40 (rabbit), or control IgG. Precipitated proteins were immunoblotted (WB) with EGFR or Tom40 antibodies. D, biochemical fractionation of the EGFR and c-Src with the mitochondria is shown. 10T1/2 cells were treated as described under “Experimental Procedures.” Fractionation yielded proteins present in and associated with the mitochondria (crude mitochondria), proteins inserted in the mitochondrial membrane (alkali-resistant), and proteins peripherally attached to the mitochondrial membrane (alkali-sensitive). Fractions containing cell equivalents were immunoblotted with the following antibodies: EGFR, c-Src, voltage-dependent anion channel (VDAC), Rab4, Nucleoporin62, Bip/Grp, and Lamp2. Crude mitochondrial fractions represent 80 times the volume of whole cell lysates (WCL) present on the immunoblot, whereas alkali-resistant and -sensitive fractions represent 26 times the whole cell lysate volume. ER, endoplasmic reticulum.