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. 2014 Jul 17;55(2):238–252. doi: 10.1016/j.molcel.2014.05.021

Figure 2.

Figure 2

Interaction of Atg16L1 with WIPI2 Requires 207–242, a Domain Not Functionally Conserved in Atg16L2

(A) Scheme of mouse Atg16L1 and deletion mutants showing the N-terminal Atg5 interacting domain (white box), a coiled-coil domain (black box), and a WD propeller-repeat domain (striped box).

(B) Untransfected (UN) or FLAG-Atg16L1 full-length (FL), 79–623, or 1–265 constructs were expressed in HEK293A cells stably expressing GFP-WIPI2b and immunoprecipitated using GFP-TRAP. Tags were visualized by immunoblotting. I, input; UB, unbound.

(C) GFP, CFP-Atg5, GFP-WIPI1a, and GFP-WIPI2b were transiently expressed in HEK293A cells. GFP-tagged proteins were isolated using GFP-TRAP and incubated with in vitro translated 35S-labeled FLAG-Atg16L1 constructs 1–265, 1–242, 1–230, and 1–207 before washing and analysis by autoradiography. Protein expression was validated by immunoblot (bottom panel).

(D) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, or GFP-WIPI2b were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 or Atg16L2. Protein complexes were immunoprecipitated using GFP-TRAP, followed by immunoblot. See also Figure S2 and Table S1.