Immunofluorescent Analysis of Lysosomal Localization of AXIN
For monitoring dynamic translocation of AXIN onto the sur-
face of lysosome in intact cells, immunofluorescent staining is
performed, with LAMP2 as a lysosomal marker.- Cells are placed on glass coverslips in 6-well dishes and cultured
to 60–80% confluence. - Aspirate the medium and fix the cells with 1 mL of 4% (v/v)
formaldehyde at RT for 20 min (seeNote 31). - Rinse the cells twice with 1 mL of PBS (RT), and permeabilize
cells with 1 mL of PBS-T for 5 min at 4C. - Rinse the permeabilized cells twice with 1 mL of PBS, and
incubate the cells with primary antibodies against AXIN and
LAMP2 overnight at 4C. - Rinse the cells for three times with 1 mL of PBS, and then
incubate the cells with Alexa-Fluor 488-conjugated anti-goat
secondary antibody and Alexa-Fluor 594-conjugated anti-rat
secondary antibody for 8 h at RT in the dark. - Wash the cells for four times with 1 mL of PBS, and then
mount the coverslip on a slide by 20μL of mountant. - Visualize the cells under a confocal microscopy. The samples
are excited with argon gas laser using a 488-nm laser line for
Alexa-Fluor 488 dye (green channel) and with HeNe gas laser
using a 594-nm laser line for Alexa-Fluor 594 dye (red chan-
nel). Confocal microscopic pictures are taken with a 63oil
objective using the confocal microscope (seeNote 32). The
colocalization percentages are analyzed by using ZEN 2010
software (Zeiss).
Determining the Complex Formation Between v-ATPase-Ragu-
lator and AXIN/LKB1-AMPK by Co-immunoprecipitation
Analysis
Cell lysis and co-immunoprecipitation analysis are carried out
as described in Rui et al. [30] with some modifications. In particu-
lar, to ensure that the membrane-associated proteins such as Ragu-
lator are adequately extracted in the lysis buffer, a series of
detergents were screened and titrated. We have found ODG as
the most adequate detergent to be included in the lysis buffer [15].- MEFs are cultured to 70–80% confluence in 15-cm dishes. To
immunoprecipitate AXIN or LAMTOR1, 4–10 dishes (15 cm)
of MEFs are collected. - Aspirate the medium and quickly lyse the cells with 750μL/
dish of ice-cold ODG buffer (seeNote 33). To immunoprecip-
itate AXIN or LAMTOR1, lyse lysosomes prepared from ten
10-cm dishes or DRMs from five 10-cm dishes with 750μL/
dish of ice-cold ODG buffer.
402 Chen-Song Zhang et al.