Amicon ultra 0.5 10 kda centrifugal filter units

The growth media of HeLa WT and HeLa TGN46 KO cells seeded on a well of 6-well plate – and transfected with PAUF-Myc-His 48 hr previous to the experiment performance – was exchanged by pre-warmed (37°C) DMEM containing 1% penicillin/streptomycin and 2 mM L-glutamine. Cells were then incubated for 4 hr in an incubator at 37°C and 5% CO₂ to collect secreted PAUF-Myc-His protein. Then, media was collected in a 1.5-ml tube and centrifuged to avoid floating dead cells that may interfere. Protease inhibitor was added to sample and sample was concentrated down to 150 µl using Amicon Ultra-0.5 10 kDa Centrifugal Filter Units (Merck). On the other hand, cells were lydes on ice with 150 µl of ice-cold lysis buffer (1% SDS Tris–HCl pH 7.4). Samples were collected in a 1.5 ml tube using a scraper and centrifuged at maximum speed for 20 min at 4°C to remove cell pellet. Finally, 6× Laemmli SDS sample buffer was added to both, supernatant and cell samples, and incubated for 10 min at 95°C. The samples were subjected to SDS–PAGE and Western blotting with anti-Myc (X) and anti-TGN46 (AbD Serotec) antibodies. When cells were treated with BFA, the compound was added to the cells to a final concentration of 5 µg/ml 15 min before media was exchanged by FBS-deprived media and also to the collection media for the 4 hr incubation time of the secretion experiment.