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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Nat Cell Biol. 2010 Jul 18;12(8):747–757. doi: 10.1038/ncb2078

Figure 1. Atg16L1 interacts with clathrin-heavy chain.

Figure 1

A. HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, subjected to SDS-PAGE, and proteins were stained with SimplyBlue safe stain (Invitrogen) according to manufacturer's protocol. Bands indicated in the box were cut out and digested with trypsin, and identified by MALDIToF. B. HeLa cells transfected with control vector, or wild-type or different deletion mutants (2-77, 2-275, 232-607) of Flag-Atg16L1 for 24h were immunoprecipitated with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Clathrin interacts with the N-terminus of Atg16L1, similar to Atg12-Atg5. Total lysates were run alongside as controls for protein input. C. HeLa cell lysates were immunoprecipitated (IP) with Atg16L1 antibody (+Ab) or no antibody (No Ab) and immunoblotted with anti-clathrin, anti-AP2 and anti-Atg16L1 antibodies. Total cell lysates (TL) were also blotted for input controls. Endogenous Atg16L1 interacts with clathrin-heavy chain and AP2. D. HeLa cells transfected with Flag-Atg16L1 and increasing concentrations of Atg5 for 24h were immunoprecipitated (IP) with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Atg5 overexpression neither interferes with, nor increases, clathrin-Atg16L1 binding. TL – total cell lysate.