- We strongly recommend that the mouse be housed individually
particularly during sacrifice. - The lysis buffer can be prepared in large batches, as 10stock
solutions and frozen in aliquots. - It is convenient to use Mini Trans-Blot®Cell from Bio-Rad
Laboratories. - For immunoblotting, the antibodies against phospho-AMPK-
α-Thr-172 (cat. #2535), anti-AMPKα(cat. #2532), anti-phos-
pho-ACC-Ser79 (cat. #3661), and anti-ACC (cat. #3662) can
be purchased from Cell Signaling Technology. All of these
antibodies are 1:1000 diluted for immunoblotting. - The T 10 basic ULTRA-TURRAX®from IKA equipped with
an S10N-5G Dispersing element (cat. 0003304000) is
suggested. - The method for the preparation of lysosome was originally
developed by Sigma-Aldrich LLC, and the materials used
here were purchased from Sigma. - To prepare the sucrose solution, dissolve sucrose at 37Cin
Buffer A. Vigorous shaking or vortexing should be avoided,
since it would make bubbles. - For IP, the antibody against LAMTOR1 was raised with pur-
ified bacterially expressed GST-tagged fragment of amino acids
1–64 of human LAMTOR1. The antibody against AXIN can
be purchased from Santa Cruz Biotechnology (cat. sc-8567,
diluted 1:100). - For immunofluorescent staining, the antibody against AXIN
can be purchased from Santa Cruz Biotechnology (cat.
sc-8567, diluted 1:60), and the antibody against LAMP2
from Abcam (ab13524, diluted 1:120). - It is convenient to use ProLong Diamond Antifade Mountant
(Molecular Probes, P36970). 90% (v/v) glycerol can also be
used as mountant. - Keep the concentration of NaCl below 100 mM; otherwise,
the efficacy of ODG will be dampened. - It is convenient to use the electrolyte containing phosphate
buffer from Human Metabolome Technologies (cat. H3302-
1022). - The size of clamp should be slightly larger than that of a
hepatic lobe. Make sure that the lobe can be tightly clamped
by the clamp. - Only glucose starvation is mentioned as a representative of
AMPK-activating stresses in this section. For starvation of
other nutrients, just choose appropriate media accordingly.
For treatment of drugs, the drug of interest can be added to
Analysis of Lysosomal AMPK Activation 407