- 1160 mm centrifuge tubes.
- Buffer A: 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 5 mMβ-mercaptoethanol, 0.25% (v/v) Triton X-100,
with protease inhibitor cocktail. Stored at 4C. - Sucrose solutions: 5%, 35%, and 80% (w/v) sucrose in Buffer A
(seeNote 10). Store at 4C. - Ultracentrifuge equipped with SW41 Ti rotor. The rotor
should be precooled to 4C prior to use. - 1489 mm centrifuge tubes.
- Microscope: confocal microscope equipped with argon gas
laser and HeNe gas laser. - Formaldehyde solutions: 4% and 8% (w/v) formaldehyde
in PBS. - Antibodies for IP: rabbit anti-LAMTOR1 and goat anti-AXIN
(seeNote 11). - Antibodies for immunofluorescent staining: goat anti-AXIN,
rat anti-LAMP2 primary antibodies (seeNote 12), and Alexa-
Fluor 488-conjugated anti-goat, Alexa-Fluor 594-conjugated
anti-rat secondary antibodies. - Mountant (seeNote 13).
- ODG buffer: 50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 2% (w/v) octylβ-D-glucopyranoside
(ODG), 5 mMβ-mercaptoethanol, with protease inhibitor
cocktail (seeNote 14). Store at 4C. - Protein A/G beads: mix rProtein A Sepharose Fast Flow with
an equal volume of Protein G Sepharose 4 Fast Flow. Store at
4 C. - PBS-T: PBS, 0.1% (v/v) Triton X-100.
- DRM buffer: 2% (w/v) ODG, 1% (v/v) Nonidet P-40
(NP-40) in Buffer A.
2.3 In Vitro
Reconstitution
of Lysosomal AMPK
Activation
- Fractionation buffer: 50 mM KCl, 90 mM potassium gluco-
nate, 1 mM EGTA, 5 mM MgCl 2 , 50 mM sucrose, 20 mM
HEPES, pH 7.4, supplemented with 2.5 mM ATP, amino
acids, and protease inhibitor cocktail. Store at 4C. - 22 G needles and 1-mL syringes.
- AMP: 100 mM AMP in H 2 O. Store at 20 C.
- ATP: 500 mM ATP in H 2 O. Store at 20 C.
- Protein phosphatase 2Ac (PP2Ac): 1μg/μL PP2Ac,50mM
Tris–HCl, pH 7.0, 14 mMβ-mercaptoethanol, 1 mM benza-
midine, 0.1 mM PMSF, 1 mM EDTA, and 50% (v/v) glycerol.
Store at 20 C.
Analysis of Lysosomal AMPK Activation 397