Figure 6.
Figure 6. PI4P and PI4KIIIβ are associated with autophagosomal structures. (A) HEK293A cells stably expressing GFP-ATG13 were incubated in ES for 2 h, fixed, and labeled using antibodies to PI4P. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. (B) HEK293A cells transfected with GFP-P4MX2 were incubated in ES for 2 h, fixed, and labeled using antibodies to LC3B and WIPI2. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. (C and D) Quantification of the percentage of GFP-ATG13 (C) or LC3 (D) spots associating with PI4P, PI4KIIIβ, and WIPI2 (analysis of 100 spots). (E) HEK293A cells transfected with GFP-P4MX2 were incubated in ES for 2 h, and the GFP-P4MX2-positive compartment was immunoisolated before immunoblot for GFP, ULK1, ATG9A, and LC3B. (F) HEK293A-GFP-ATG13 cells were incubated in ES for 2 h, fixed, and labeled using antibodies to PI4KIIIβ and WIPI2. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. In A, B, and F, arrowheads indicate colocalized structures. (G) HEK293A cells stably expressing GFP-LC3B, GFP-ATG13, or GFP-DFCP1 were incubated in ES for 2 h, and the GFP-positive compartment was immunoisolated before immunoblot for GFP, ULK1, PI4KIIIβ, and pS294 PI4KIIIβ. (H) Quantification of the relative amount of ULK1, PI4KIIIβ, and pS294 PI4KIIIβ in GFP-ATG13- and GFP-DFCP1–positive compartment. Mean ± SEM, n = 3 experiments.

PI4P and PI4KIIIβ are associated with autophagosomal structures. (A) HEK293A cells stably expressing GFP-ATG13 were incubated in ES for 2 h, fixed, and labeled using antibodies to PI4P. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. (B) HEK293A cells transfected with GFP-P4MX2 were incubated in ES for 2 h, fixed, and labeled using antibodies to LC3B and WIPI2. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. (C and D) Quantification of the percentage of GFP-ATG13 (C) or LC3 (D) spots associating with PI4P, PI4KIIIβ, and WIPI2 (analysis of 100 spots). (E) HEK293A cells transfected with GFP-P4MX2 were incubated in ES for 2 h, and the GFP-P4MX2-positive compartment was immunoisolated before immunoblot for GFP, ULK1, ATG9A, and LC3B. (F) HEK293A-GFP-ATG13 cells were incubated in ES for 2 h, fixed, and labeled using antibodies to PI4KIIIβ and WIPI2. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. In A, B, and F, arrowheads indicate colocalized structures. (G) HEK293A cells stably expressing GFP-LC3B, GFP-ATG13, or GFP-DFCP1 were incubated in ES for 2 h, and the GFP-positive compartment was immunoisolated before immunoblot for GFP, ULK1, PI4KIIIβ, and pS294 PI4KIIIβ. (H) Quantification of the relative amount of ULK1, PI4KIIIβ, and pS294 PI4KIIIβ in GFP-ATG13- and GFP-DFCP1–positive compartment. Mean ± SEM, n = 3 experiments.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal