- In our experience, the antibodies listed in Subheading2.5
worked well, which is why we share the supplier information.
However, it does not preclude that antibodies recognizing the
same proteins or modifications obtained from other suppliers
could also be used in this protocol. - If the increase or decrease of a phosphorylation signal should
be evaluated in response to a certain treatment, the total level of
protein needs to be detected to be able to evaluate the ratio of
phosphorylation to total protein. This proceeding excludes the
misinterpretation due to possible effects on the total protein
level. - To evaluate an influence on the AMPK-dependent induction of
autophagy, the successful AMPK activation should always be
controlled to exclude side effects or misinterpretation. This can
be realized by the detection of the AMPK-mediated Ser79
phosphorylation of ACC that is part of an autophagy-
independent pathway. The activating phosphorylation of
AMPK itself, namely, Thr172, can be utilized for the same
purpose but is less reliable, especially with regard to AICAR
and A-769662 treatment (seeFig. 1a and b)[ 4]. - In combination with the degradation of p62, another marker
for the induction of autophagy should be evaluated, since the
p62 level depends on the experimental setup. During longer
periods of starvation or after protein overexpression, p62 is
transcriptionally upregulated. This effect abolishes the short-
term degradation of p62 seen after autophagy induction (see
Figs.2c and 3a)[ 37, 39]. - ULK1 phosphorylation level as well as the total protein level
change in response to the AMPK-dependent induction of
autophagy depending on the temporal and cell type context
(seeFig. 2c)[ 40, 41]. Therefore an evaluation of the total
protein levels is crucial (seeNote 3). - Digitonin solution might form precipitates during storage at
4 C, which can be resolubilized by warming up to 37C and
vortexing. - To detect LC3 dots by immunofluorescence, it is absolutely
required to use digitonin for cell permeabilization since other
detergents are too harsh and destroy the autophagosomal
membranes. - It is crucial to perform the last washing steps with H 2 O and to
dilute the Hoechst dye in water since PBS would crystalize in
combination with Mowiol. - The mounting can be facilitated by pre-warming the cold
Mowiol solution to room temperature and cutting off the
plastic pipette tip to transfer the viscous solution. Not more
386 Sarah Krieg et al.