2 µg of respective recombinant Arabidopsis thaliana ATG8 isoform, denatured at 95C for 2 min. were separated on % SDS-PAGE and blotted 1h to nitrocellulose (GE10600003) using semi-dry transfer. Blots were blocked with 3% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 4 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:5 000 in for 1h at RT with agitation. The blot was washed as above and developed on film (GE28-9068-37) with high sensitivity chemiluminescent detection reagent. Exposure time was 1 second.
Courtesy of Dr. Steingrim Svenning, UiT, The Arctic University, Norway
Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 29079776
Journal: Nat Commun
Figure Number: 5A
Published Date: 2017-10-27
First Author: Dong, Y., Silbermann, M., et al.
Impact Factor: 13.783
Open Publication
Autophagy is specifically induced by limited S-precursor supply for cysteine biosynthesis. a Autophagy induction in the shoot of serat tko and sir1-1 was determined by immunological detection of the canonical autophagy marker ATG8a and NBR1 with specific antisera. Lipidation of ATG8a (ATG8a-PE) (apparent size: 15–20?kDa) is essential for autophagosome formation and indicated by a significant shift during electrophoresis. NBR1 is a cargo receptor for selective autophagy and consequently degraded in autophagic bodies (D-NBR1) (apparent size: 50–75?kDa). b Level of ATG8a-PE and D-NBR1 shown in a were quantified (n?=?3, mean?±?s.e.m., one-way ANOVA, *p?
Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 29079776
Journal: Nat Commun
Figure Number: 5D
Published Date: 2017-10-27
First Author: Dong, Y., Silbermann, M., et al.
Impact Factor: 13.783
Open Publication
Autophagy is specifically induced by limited S-precursor supply for cysteine biosynthesis. a Autophagy induction in the shoot of serat tko and sir1-1 was determined by immunological detection of the canonical autophagy marker ATG8a and NBR1 with specific antisera. Lipidation of ATG8a (ATG8a-PE) (apparent size: 15–20?kDa) is essential for autophagosome formation and indicated by a significant shift during electrophoresis. NBR1 is a cargo receptor for selective autophagy and consequently degraded in autophagic bodies (D-NBR1) (apparent size: 50–75?kDa). b Level of ATG8a-PE and D-NBR1 shown in a were quantified (n?=?3, mean?±?s.e.m., one-way ANOVA, *p?<?0.05). c Autophagy induction in the root of serat tko and sir1-1 was detected by MDC staining. White arrows marked the visible autophagic bodies. Scale bar, 25?µm. d Autophagy induction in the shoot of WT under sulfur deficiency was determined by ATG8a-PE level (n?=?3, mean?±?s.e.m., t-test, *p?<?0.05)
