In Vitro Harvest

Following designated treatment, wash cells three times with DMEM followed by the application of 0.5mL of homogenization buffer supplemented with proteinase and phosphatase buffer.

Homogenisation Buffer:

A	B
Sucrose	250 mM
EDTA	2 mM
Magnesium chloride	1.5 mM
Potassium Chloride	10 mM
HEPES	20 mM

Gently detach cells with cell scraper.

Homogenize using Teflon homogenizer (12 strokes).

Separate 50µL of total homogenate (TH) and lyse with *TNE lysis buffer.

TNE lysis buffer:

A	B
Tris	50 mM
NaCl	150 mM
EDTA, pH 7.4	5 mM
SDS	1%
NP-40	0.5%
DOC	0.5%
protease/phosphatase inhibitors

Centrifuge remaining volume of TH at 1000x g,4°C, collect the supernatant.

Further centrifuge the supernatant for 20000x g,4°C to collect the precipitate as crude lysosomal fraction (CLF). Lyse the CLF with *TNE lysis buffer, and prepare lysates for western blot.

TNE lysis buffer:

A	B
Tris	50 mM
NaCl	150 mM
EDTA, pH 7.4	5 mM
SDS	1%
NP-40	0.5%
DOC	0.5%
protease/phosphatase inhibitors