Transient Transfection, Protein Extraction and Cell Manipulations

Transient transfections of HEK293 or HeLa cells were conducted at 60–70% confluency using MACSfectin Reagent (130-098-412, Miltenyi Biotec). For TETexpress-inducible system, transactivator was added 24 h after transfection following manufacture instructions (Clontech). At indicated times, medium was removed and stored at −80°C in presence of protease inhibitors (Roche, 11697498001); cells were collected and washed three times with PBS before addition of lysis buffer (300 mM NaCl, 50 Mm NaH₂PO₄ pH 8, 1% Triton X-100) or RIPA buffer (25 mM Tris/HCl 150 mM NaCl, pH 7.6, 1% NP - 40, 1% sodium deoxycholate, 0.1% SDS) as indicated, both containing protease inhibitors cocktail. Cell lysis was done for 30 min on ice followed by centrifugation at 21.000 × g at 4°C. Protein fraction was collected from the supernatants and immediately processed for immunoprecipitation or stored at −80°C for further analysis. Protein concentrations were determined using Bradford (Bio-Rad, 5000006) or BCA (Bio-Rad, 23225) colorimetric assays. Absorbance was measured with Varioskan Flash (Thermo Fisher Scientific). Conditioned media was also collected and proteasome inhibitors cocktail added. For acetone precipitations, 4 h before collection, conditioned medium was removed and cells were carefully washed three times with pre-warmed serum-free medium (Lonza, 12–764Q) supplemented with 2 mM L-glutamine. Serum-free medium (SFM) was added and left for that period in culture conditions. Four volumes of ice-cold acetone were added to the medium, mixed and incubated for 15 min on ice. Samples were centrifuged at 12,000 × g for 10 min at 4°C. Supernatants were carefully removed and precipitates were resuspended in loading buffer or in water for enzymatic treatment. Proteasome inhibition: Protein degradation was studied as published (32). Briefly, 24 h after transfection a mixture of proteasome inhibitors (5 μM lactacystin, 5 μM MG132, and 1 mM epoxomycin) was added and 18 hrs later, cells were harvested, lysed and analyzed by immunoblotting. Cell fractionation: Cell fractionation was carried out according to Holden and Horton protocol (33).