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1.
FIGURE 2.

FIGURE 2. From: Mitophagy in Yeast Occurs through a Selective Mechanism.

Monitoring mitophagy using C-terminal GFP-tagged mitochondrial protein processing at post-log phase. Wild-type (WT), atg1Δ, and pep4Δ strains expressing Om45-GFP or Idh1-GFP were cultured in YPL medium for 1–3 days. The localization of GFP was visualized by fluorescence microscopy at day 2 (A), and GFP processing was monitored by immunoblotting with anti-GFP and anti-Pgk1 (loading control) antibody and antiserum, respectively, at the indicated days in the strains expressing Om45-GFP (B) or Idh1-GFP (C). DIC, differential interference contrast.

Tomotake Kanki, et al. J Biol Chem. 2008 November 21;283(47):32386-32393.
2.
FIGURE 1.

FIGURE 1. From: Mitophagy in Yeast Occurs through a Selective Mechanism.

Monitoring mitophagy using C-terminal GFP-tagged mitochondrial protein processing during amino acid starvation. Wild-type (WT), atg1Δ, or pep4Δ strains expressing Om45-GFP (A) or Idh1-GFP (B) were cultured in YPL medium to mid-log growth phase and then shifted to SD-N medium for 0, 4 and 6 h. GFP processing was monitored by immunoblotting with anti-GFP and anti-Pgk1 (loading control) antibody or antiserum, respectively. The positions of molecular mass markers are indicated on the right.

Tomotake Kanki, et al. J Biol Chem. 2008 November 21;283(47):32386-32393.
3.
FIGURE 4.

FIGURE 4. From: Mitophagy in Yeast Occurs through a Selective Mechanism.

Screening atg mutants for potential mitophagy defects during starvation. Strains deleted for the indicated ATG genes and expressing Om45-GFP were screened for potential mitophagy activity using the method demonstrated in Fig. 1. The atg mutant strains in A are either defective in all types of autophagy (atg8Δ and atg9Δ) or are defective in the Cvt pathway. The atg mutant strains in B are defective for all types of autophagy (atg1Δ) or are defective in nonspecific macroautophagy, but are normal for the Cvt pathway. The relative amount of processed GFP was calculated. The values represent the means and standard deviation from three independent experiments. WT, wild type.

Tomotake Kanki, et al. J Biol Chem. 2008 November 21;283(47):32386-32393.
4.
FIGURE 6.

FIGURE 6. From: Mitophagy in Yeast Occurs through a Selective Mechanism.

Analysis of pexophagy and the Cvt pathway. Wild-type (WT) and atg11Δ, atg20Δ, and atg24Δ (A) or atg17Δ, atg29Δ, and atg31Δ (B) strains expressing Pex14-GFP were cultured with oleic acid-containing medium for 19 h, then shifted to SD-N for the indicated times, and monitored for GFP processing by immunoblotting. The asterisks indicate nonspecific bands. The relative amount of processed GFP was calculated. The values represent the means and standard deviation from three independent experiments. C, each mutant was cultured in YPD medium and analyzed for prApe1 maturation by immunoblotting to monitor the Cvt pathway during vegetative growth. The positions of precursor and mature Ape1 are indicated.

Tomotake Kanki, et al. J Biol Chem. 2008 November 21;283(47):32386-32393.
5.
FIGURE 5.

FIGURE 5. From: Mitophagy in Yeast Occurs through a Selective Mechanism.

Screening atg mutants for mitophagy defects during post-log phase growth. Strains deleted for the indicated ATG genes and expressing Om45-GFP were cultured in YPL medium for 2 days. A, cells were collected at the indicated days and monitored for GFP processing by immunoblotting. The relative amount of processed GFP was calculated. The values represent the means and standard deviation from three independent experiments. B, the indicated strains cultured in YPL for 2 days were observed by fluorescence microscopy. The phenotype of the atg8Δ strain was essentially identical to that of atg9Δ, and that of atg20Δ was essentially identical to that of atg24Δ. DIC, differential interference contrast. WT, wild type.

Tomotake Kanki, et al. J Biol Chem. 2008 November 21;283(47):32386-32393.
6.
FIGURE 3.

FIGURE 3. From: Mitophagy in Yeast Occurs through a Selective Mechanism.

Macroautophagy occurs independent of mitophagy. Wild-type (WT) cells expressing Idh1-GFP (A) or GFP-Atg8 (B) were cultured in YPL medium for 12 h and then starved in SD-N or SL-N for up to 6 h. The cells were collected at the indicated time points, and cell lysates equivalent to A600 = 0.1 unit of cells were subjected to immunoblot analysis with anti-GFP and anti-Pgk1 (loading control) antibody or antiserum, respectively. C, the Pho8Δ60 strain (TN124) expressing Om45-GFP was cultured in YPL medium for 12 h and starved in SD-N or SL-N for 6 h. Cell lysates equivalent to A600 = 0.1 unit of cells were subjected to immunoblot analysis as above. D, cell lysates equivalent to A600 = 2 units of cells were analyzed by the Pho8Δ60 activity assay. The Pho8Δ60 activity of wild-type cells starved in SD-N was set to 100%. The values represent the mean and standard deviation from three independent experiments. The Pho8Δ60 activity of the atg1Δ strain cultured in YPL is shown to indicate the background activity of this assay.

Tomotake Kanki, et al. J Biol Chem. 2008 November 21;283(47):32386-32393.

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