- The transfection immediately after seeding the cells saves 1 day
and increases the transfection efficiency. - The time necessary to achieve an efficient knockdown depends
on the protein and its half-life. In this case, a knockdown for
72 h is sufficient for the AMPKα-subunits (seeFig. 2c). Addi-
tionally, an efficient knockdown was also possible using the
calcium-phosphate precipitation method, which further
enabled co-transfection of desired plasmids. - A curved/bent forceps facilitates the handling of the coverslips
(e.g., Roth, Dumont #7, 11274-20). - Equal amounts of proteins have to be loaded to compare
treated with untreated cells with regard to LC3 conversion as
well as phosphorylation signals to allow an assessment of the
induction of autophagy. This does not substitute the detection
of a loading control or total protein levels. - To evaluate the conversion of the LC3-I to the
autophagosome-associated LC3-II form, the lysates have to
be fresh, i.e., not older than 2 or 3 days, since the fragile
LC3-II form is prone to degradation. - The larger proteins like Raptor (150 kDa), ULK1 (150 kDa),
and ACC (280 kDa) are best visualized with the help of a 7.5%
SDS gel and a tank blot to facilitate their transfer onto the
nitrocellulose membrane. - To evaluate the conversion of the LC3-I to the LC3-II form by
immunoblotting, a 15% SDS polyacrylamide gel needs to be
employed to visualize the difference in size due to proteolytic
cleavage and lipidation [18, 19]. - TBS-T should be used instead of PBS-T when phosphorylation
signals should be detected. - If the signal is weak, the sensitivity of the ECL solution can be
enhanced by using a more sensitive variant of the ECL sub-
strate solution. - Stripping the blot to detect the total protein level after detect-
ing the phosphorylation of the same protein or vice versa is not
recommended, since stripping might falsify the results due to
unequal co-removal of proteins from the membrane. - When the induction of autophagy should be assessed, the
LC3-II form is the main readout parameter. Therefore a densi-
tometry analysis of LC3-II in relation to a loading control like
actin or tubulin can serve as a quantifiable measure for the
amount of autophagy in the cells (seeFig. 1a and b)[ 25]. - In immunofluorescence analysis, the usage of relatively large
and strongly adherent cells is recommended in order to have
rather large cytoplasmic area that can be recorded in a single
plane for the quantification of autophagosomes.
388 Sarah Krieg et al.