AMPK Methods and Protocols

33. Sonicate the lysates (see Note 34), and centrifuge at
    20,000gat 4C for 15 min.


34. Incubate the centrifuged supernatants with antibody against
    AXIN or LAMTOR1 overnight on a rotator at 40 rpm at 4C.


35. Overnight protein aggregates are precleared by centrifugation
    at 20,000gat 4C for 10 min, and protein A/G beads are
    added into the supernatant into the 7.5 mL of supernatant and
    mixed on a rotator at 40 rpm for 3 h at 4C. Some 75 and
    30 μL of net volumes of beads are needed to immunoprecipi-
    tate AXIN and LAMTOR1, respectively.


36. Wash the beads with 100 volumes of ODG buffer for three
    times at 4C. Aspirate the ODG buffer, mix the beads with an
    equal volume of 2SDS sample buffer, and boil the mixture
    for immunoblotting (described in steps 7– 11 of
    Subheading3.1).

3.3 In Vitro
Reconstitution
of Lysosomal AMPK
Activation

In this section, the methods for the purification of light organelles,

and the versatile applications of these membrane structures in

monitoring the binding of AXIN/LKB1 to lysosome and activation

of AMPK, are described.

Purification of Light Organelles

1. Treat MEFs with desired stimuli.


2. Scrape and spin down cells at 200gat RT, and then resus-
   pend the cells in 500μL per 10-cm dish of fractionation buffer
   at RT.


3. Mechanically break the cells by passing each cell resuspension
   through a 22 G needle attached to a 1-mL syringe for six times
   (see Note 35), and then spin down the homogenates at
   2000 gfor 10 min, yielding PNS.


4. The PNS is then spun at 20,000gfor 15 min at 4C. The
   pellets are light organelles, and the supernatants are
   membrane-cleared cytosolic fractions.

In Vitro Reconstitution Assay for Lysosomal Association of

AXIN/LKB1

To recapitulate starvation-induced translocation of AXIN onto

the surface of lysosome, we incubated light organelles purified from

glucose-starved or unstarved cells with AXIN in the membrane-

cleared cytosols or purified bacterially expressed AXIN [30]. We

found that after glucose starvation or v-ATPase inhibition, the v-

ATPase-Ragulator complex becomes more accessible to AXIN-

LKB1 [15]. Similarly, the effects of other metabolic stresses or

pharmacological stimuli on the lysosomal v-ATPase-Ragulator

complex can be easily determined in vitro through applying this

method.

Analysis of Lysosomal AMPK Activation 403