KFERQ motif is required for EGFR degradation through CMA upon oz-apraA treatment. (A) Sequence alignment of the EGFR family members from human and mouse. The αC helix contains a ₇₅₆NKEIL₇₆₀ pentapeptide sequence (yellow box), which is known as a KFERQ motif recognized by CMA. Pink regions indicate the homogeneity of the amino acid between EGFR family proteins, and purple and red regions indicate the discrepancy between ErbB3 and other members. N, Asn; −, basic amino acid; +, acidic amino acid; Φ, hydrophobic amino acid; *, Hsp90-binding sites. (B) HEK293 cells were transfected with wild-type (WT) EGFR or EGFR(AA) for 24 h and stimulated with 100 ng/ml EGF for 5 min. The levels of selected EGFR downstream protein phosphorylation were detected using specific anti–p-STAT1, anti–p-STAT3, and anti–p-EGFR(Y1068) antibodies. Anti-GAPDH was used as a loading control. Black lines indicate that intervening lanes have been spliced out. (C) The treatment of oz-apraA induces the degradation of EGFR(AA) through MG132-sensitive proteasome pathway. HEK293 cells were transfected with wild-type EGFR or EGFR(AA) for 24 h and treated with 100 nM oz-apraA, 5 µM MG132, or 20 mM NH₄Cl for an additional 24 h. The cells were lysed, and the degradation of EGFR was analyzed using anti-EGFR by Western blotting. Anti–α-tubulin was used as a loading control.