Fig. 3. Two sites in LC3 are essential for p62 binding. HeLa cells were transfected with the gene of interest and after 24 hours were cultured for 2 hours in EBSS medium in the presence of 0.1 µM Baf A. Cells were then lysed using RIPA extraction buffer. GFP or GFP fusion constructs of LC3 and its mutants (A) or LC3 family proteins and their mutants (B) were immunoprecipitated (IP) from total cellular extract (500 µg) and subjected to SDS-PAGE. Copurified p62 was detected by immunoblotting with anti-p62 antibody (upper panel). WB, western blot. Immunoprecipitated GFP fusion proteins were detected with anti-GFP antibody (lower panel). Amount of total p62 or GFP fused proteins detected in 50 µg total protein extract (extracts) is indicated.