Membrane and cytosol fractionation

Cells (one 10 cm dish) were cultured to 70% confluence and transfected with different constructs

of DNAJC5

One day after transfection, we harvested the transfected cells by scraping in 1 ml B88 (20 mM HEPES-KOH, pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, and 5 mM magnesium acetate) plus a cocktail of protease inhibitors

Cells were homogenized by 10 passages through a 22G needle

Homogenates were centrifuged at 500×g for 0h 10m 0s and the resulting post-nuclear supernatant (PNS) fractions were centrifuged at 100,000×g for 1h 30m 0s

High-speed supernatant fractions were then subjected to a repeat centrifugation to achieve a clarified cytosol fraction

The pellet fraction was washed and resuspended in the same volume of B88

Resuspended material was also centrifuged again to collect a washed membrane fraction

Membranes were lysed in lysis buffer

The PNS was subjected to differential centrifugation at 3000×g for 0h 10m 0s

The supernatant was centrifuged at 25,000×g for 0h 20m 0s

The supernatant was centrifuged at 100,000×g for 0h 30m 0s

Membrane fractions were normalized to phosphatidylcholine content and analyzed by immunoblot

The 25,000×g membrane fraction was aliquoted into three tubes: one without proteinase K, one with proteinase K (10 μg/ml), and one with proteinase K plus TritonX-100 (0.5%)

The incubation was conducted On ice for 0h 20m 0s

The reaction was stopped by sequential addition of PMSF (1 mM)

Add sample buffer. Then, samples were then heated on metal block at 95°C for 0h 5m 0sand analyzed by SDS-PAGE and immunoblot.