- Rinse cells twice with 2 mL of PBS (RT).
- Add 2 mL of complete DMEM medium or glucose-free
DMEM supplemented with desired concentrations of glucose
into the control cells or cells to be starved. Incubate cells for
desired periods of time at 37C in the CO 2 incubator. - Aspirate medium, and quickly add 250μL of ice-cold lysis
buffer into each well, and then place the dishes on ice (see
Notes 20and 21 ). - Scrape and collect the lysed cells, and then sonicate for 3–5 s at
30% maximum power on ice (seeNote 22). The lysates are then
centrifuged at 20,000gfor 10 min at 4C. - Transfer 250μL of each supernatant into a new tube contain-
ing 250μLof2SDS sample buffer, mix well, and then boil
the mixture for 10 min. - Load 10μL of each sample into the wells of an 8% SDS-PAGE
gel, along with molecular weight marker. Run the gel for 1–2 h
at 100 V. - Activate PVDF with methanol for 1 min and transfer the
protein from the gel to the PVDF membrane by a vertical
“wet” transfer apparatus at 100 V for 1 h. - Block the membrane for 1 h at room temperature using block-
ing solution containing 5% (w/v) nonfat dry milk (for blocking
AMPKαand ACC) or 5% (w/v) bovine serum albumin (for
blocking p-AMPKαand p-ACC). - Incubate the membrane with primary antibody in blocking
solution overnight at 4C, and then incubate the membrane
with the HRP-conjugated secondary antibody in blocking
solution at RT for 1 h. - Acquire images using techniques for chemiluminescence.
Starvation of Mice and Determination of p-AMPKα-Thr-172 in
the Mouse Liver
- Withdraw the diet at 5 p.m.
- Sacrifice mice at 9 a.m. the next day by cervical dislocation (see
Note 23). Liver tissues are excised and instantly frozen in
liquid nitrogen (seeNote 24). - To begin homogenization, thaw the frozen liver tissues on ice
and add 10μL of ice-cold lysis buffer per mg of liver tissue (see
Note 25). - Homogenize the tissues using an electrical disperser, and soni-
cate the lysates on ice for 5 s at 30% maximum power on ice. - Centrifuge the lysates at 20,000gfor 10 min at 4C, recover
supernatant in a new tube and add an equal amount of 2SDS
sample buffer, and then analyze p-AMPK and p-ACC as
described in previoussteps 6– 11.
Analysis of Lysosomal AMPK Activation 399