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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Nat Cell Biol. 2014 Apr 6;16(5):401–414. doi: 10.1038/ncb2934

Figure 1. Atg16 associates with Cx43 in an early step of autophagosome biogenesis.

Figure 1

(a) Immunoblot for the indicated Cx of homogenates and autophagic vacuoles (AV) isolated from fed or 6h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a. (b) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 maintained in the presence or absence of serum for 4h. Single channels are shown in reverse black and white and magnification of single and merged images are shown in color. Arrows: colocalization of Atg16/Cx43 (yellow) or Atg16/Cx43/LC3 (white). Quantification is shown in Supplementary Fig. 1c. (c) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4h. Full fields are shown in Supplementary Fig. 1d. (d) Maximum projection confocal microscopy 3D reconstruction (left) followed by a single plane time-lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2-Cx43 (pseudocolored in red) and sfGFP-ATG16. (e) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white and merged images in color. Inset shows higher magnification. Bottom: Quantification of colocalization between Atg16 and Cx43 (n=3 wells, 4 independent experiments, >20 cells per experiment). Values are mean+s.e.m. (*) p <0.05. (f, g) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 and Atg16 from mouse embryonic fibroblasts (MEF) wild type cells maintained in the presence (f, g) or absence of serum (4h) (g). I: input; FT: flow through. (h) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 from MEFs wild type untreated (Wt), upon incubation with 3-methyladenine (3MA) or in MEFs from Atg5 null mice (Atg5−/−). (i) Immunoblot for flag and Cx43 of immunoprecipitates (IP) of endogenous Cx43 in Wt MEFs transfected with the indicated flag-tagged constructs of truncated Atg16. GAPDH, LC3 and Atg5 are show as negative controls for IP. All values are mean+s.e.m. Nuclei are highlighted with DAPI. Bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.