MKO () cells transformed with different combinations of plasmids were grown in selective SMD medium, collected at mid-log phase or 2 h after starvation, and then subjected to Western blot analysis using anti-Atg8 antiserum. 0.2 OD units of cells were loaded in each lane. Pgk1 was used as a loading control. Plasmids expressing Atg8 (pATG8(414)); Atg8, Atg4, Atg7, and Atg10 (pATG8-ATG4-ATG7-ATG10(414)); Atg5, HA-Atg12, and Atg16 (pATG5-HA-ATG12-ATG16(416)); and Atg8ΔR, Atg7, and Atg10 (pATG8ΔR-ATG7-ATG10(414)) were used as indicated. Atg8–PE was hardly detected in both growing and starvation conditions even when all the known components from the Atg8–PE and Atg12–Atg5 conjugation systems were expressed (lane 4). However, when Atg8ΔR was expressed and Atg4 was absent, a significant amount of Atg8–PE was observed (lane 5), and the amount was further increased when all the components from the Atg12–Atg5 conjugation system were also expressed (lane 6). Note that Atg8–PE migrates aberrantly during SDS-PAGE and runs lower than Atg8. (B) The role of the Atg12–Atg5 conjugation system on Atg8–PE formation. The experimental procedures were the same as in A. Plasmids expressing Atg8ΔR (pATG8ΔR(414)); Atg8ΔR, Atg4, Atg7, and Atg10 (pATG8ΔR-ATG4-ATG7-ATG10(414)); Atg8ΔR, Atg7, and Atg10; Atg5 (pATG5(416)); HA-tagged Atg12 (pHA-ATG12(416)); Atg16 (pATG16(416)); Atg5 and HA-Atg12 (pATG5-HA-ATG12(416)); and Atg5, HA-Atg12, and Atg16 were used as indicated. The strain transformed with the plasmid expressing Atg8ΔR (pATG8ΔR(414)) and the strain were used as controls (lanes 10 and 11). When Atg4 was present, an Atg8–PE band was not detected (compare lanes 3 and 4). Expression of Atg5, Atg12, or Atg16 alone did not improve Atg8–PE formation (compare lanes 5–7 to lane 4). When the Atg12–Atg5 conjugate was formed through the expression of Atg7, Atg10, Atg12, and Atg5, the efficiency of Atg8–PE formation was greatly enhanced (lane 8). Atg16 further facilitated Atg8–PE conjugation and/or enhanced the stability of the conjugate (lane 9).Copyright information:Taken from "In vivo reconstitution of autophagy in "The Journal of Cell Biology 2008;182(4):703-713.Published online 25 Aug 2008PMCID:PMC2518709.