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. 2013 Jan 31;9(1):e1003245. doi: 10.1371/journal.pgen.1003245

Figure 1. Reduced TORC1 activity by nitrogen starvation is partially recovered in an autophagy-dependent manner.

Figure 1

(A) ATG2 (AMY182-10C) and Δatg2 (AMY182-10A) cells grown in SCD-Ura-Trp medium at 30°C were transferred to SD-N medium for the indicated times. Cell lysates were prepared using the alkaline-trichloroacetic acid method and analyzed by immunoblot with anti-GFP, anti-Atg13 and anti-Pgk1 antibodies. Progression of autophagy was monitored by accumulation of the free GFP moiety from the GFP-Atg8 fusion protein [65]. Pgk1 was used as a loading control. Uncropped images of blots are shown in Figure S5. Samples taken at 4-h intervals for 20 h demonstrated that the re-phosphorylation of Atg13 increased monotonically (data not shown). (B) ATG2 (AMY182-10C) and Δatg2 (AMY182-10A) cells grown in SCD-Ura-Trp medium at 30°C were transferred to SD-N medium for the indicated times. Total RNA was extracted and analyzed for the expression of RPS26A, RPL9A and NOG1 (left panel), and MEP2 and GAP1 (right panel) by RT-qPCR. Each sample was calibrated by TUB1 (Figure S1). (C) WT (AMY182-10C), Δatg1 (AMY236), Δatg7 (AMY237), Δatg11 (AMY238), and Δpep4 (AMY239) cells grown in SCD-Ura-Trp medium at 30°C were transferred to SD-N medium for the indicated times. Cell lysates were prepared and analyzed by immunoblot as described in (A). Uncropped images of blots are shown in Figure S5. (D) ATG1 (SAY122) and Δatg1 (AMY240) cells grown in SCD-Ura-Trp medium at 30°C were transferred as described in (C). Total RNA was extracted and analyzed for the expression of RPS26A and RPL9A (left panel) and MEP2 (right panel) by RT-qPCR. Each sample was calibrated by TUB1.