- Thereafter wash the cells attached to the dish with ice-cold PBS
and lyse in 400μl (10 cm dish) or 150μl (6 cm dish) of
Frackelton buffer (seeNote 1). Perform all subsequent steps
on ice or at 4C to prevent degradation. - After incubation for 10 min, scrape the adherent cells from the
dishes and transfer into 1.5 ml reaction tubes. - Clear lysates by centrifugation at 16,000gfor 30 min at
4 C. - Measure the protein concentration by Lowry assay according
to the manufacturer’s instruction in a 96-well plate format (see
Note 26).
ABaf. A1AICAR+A769ULK1135 P-ULK1 (Ser555)135−−+
−−+
++ACCP-ACC (Ser79)AMPKP-AMPK (Thr172)LC3-Ip62actinLC3-II24524563
63
634817Raptor(^180) P-Raptor (Ser792)
180
B
A+A
Baf. A1
A+A
Baf. A1LC3-II/actin: 1.00 3.7917.19
P-ULK1/ULK1:30.16
1. 00 29. 70 1. 6 3. 37Fig. 3Measuring the integrity of the autophagy flux with bafilomycin A1 and a double-tagged mCherry-EGFP-
LC3 construct. (a) NIH3T3 cells stably expressing the mCherry-EGFP-LC3 fusion protein were treated with
AICAR and A-769662 (AþA) and bafilomycin A1 (Baf. A1) as indicated. AMPK activity was evaluated by
assessing an increase in ACC, ULK1, and Raptor phosphorylation levels by immunoblot analysis. Autophagy
induction was monitored by an increase of endogenous LC3-II. The higher amount of LC3-II in the presence of
the lysosomal inhibitor Baf. A1 (in comparison to the AþA treatment) indicates an intact autophagic flux after
AMPK activation by AþA. (b) Representative confocal images of tandem mCherry-EGFP-LC3 expression in
NIH3T3 cells showing an overlay of the EGFP and mCherry staining signal. The activation of AMPK by AþA
leads to the formation of autophagosomes (yellowdots) and acidic autolysosomes (reddots). The Baf. A1
treatment blocks the fusion between autophagosomes and lysosomes resulting in an increase of only
autophagosomes (yellowdots) after inducing autophagy by AMPK activation by AICAR and A-769662. Scale
bar, 10μm
382 Sarah Krieg et al.