Fig. 2. LC3 specifically interacts with p62 in an N-terminus-dependent manner. (A) CHO cells stably expressing GFP-LC3 or GFP-LC3^N28 were pulsed with [³⁵S]methionine (40 mCi/ml), cultured for 2 hours in MEM (control) or EBSS medium (starvation) in the presence of 0.1 µM Baf A and lysed using RIPA extraction buffer. GFP-LC3 or GFP-LC3^N28 proteins were immunoprecipitated from 500 µg total cell extracts using 0.5 µl anti-GFP antibodies, and coprecipitated proteins were determined by autoradiography. (B) Cell extracts (3.5 mg) prepared from GFP-LC3-expressing cells were precipitated using 3.5 µl anti-GFP antibodies. Samples were separated on 12% SDS-PAGE and stained using silver staining technique. M, Marker; E, eluate; T, total; F, flow through. The interacting protein (boxed band) was identified by mass spectrometry (MS). (C) Amino acid sequence of rat p62/SQSTM1. Amino acids identified by mass spectrometry are in red. (D) p62 copurified with GFP-LC3 was detected with specific monoclonal anti-p62 antibodies.