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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 6. Atg14 is recruited to Cx43-enriched plasma membrane regions during nutritional deprivation.

Figure 6

(a) Immunoblot for the indicated proteins in homogenates and purified plasma membrane from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2+0.2), Beclin-1 (0.7+0.5), Vps34 (0.9+0.1), Atg16 (1.1+0.3), Atg14 (2.5+0.2) and only Atg14 were significant for p<0.01. (b) Immunoblot of Cx43 immunoprecipitates (IP) in mouse embryonic fibroblasts (MEFs) maintained in the presence or absence of serum. I: input; FT: flow through. GAPDH is shown as negative control for IP. (c) Immunoblot of Cx43 IP in MEFs from Wt or Atg5−/− mice grown in serum-supplemented media. (d) Immunoblot of Cx43 IP in Wt or Atg16 knockdowns (KD) MEFs maintained in the presence/ absence of serum for 4h. (e) Co-staining for Atg14 and Cx43 in NRK cells maintained in presence/absence of serum. Merged channels are shown and single channels and broad field images are shown in Supplementary Fig. 5d. Insets: higher magnification. (f) Maximum intensity confocal microscopy 3D reconstruction in HeLa cells expressing EBFP2-Cx43 (pseudocolored in red) and EGFP-ATG14 followed by a single-plane time-lapse acquisition of the boxed area; cells were incubated in serum-deprived medium for 1h before imaging. (g) Immunoblot of Atg14 IP in Wt or Atg5−/− MEFs maintained in the presence of serum. (h) Immunoblot in total homogenates and plasma membrane (PM) purified from Wt or Cx43−/− mice. (i) Immunofluorescence for e-cadherin and Vps34 in NRK cells wild-type (Wt) or knocked down (−) for Cx43. Single channel and merge (left) and Z-stack surface reconstruction and higher magnification of the boxed region (right). (j) Immunostaining for Cx43 and e-cadherin in NRK cells expressing a tandem FYVE domain (2xFYVE) tagged to GFP and maintained in serum-free media for 4h. Single channels (inverted black and white) and the merged image of the boxed area (color). Inset: higher magnification image. Bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.