Nif1317

Western blot was performed as described previously [12 (link)]. Briefly, total (25 µg), cytoplasmic (25 µg) or nuclear (30 µg) proteins were separated by SDS-PAGE under reducing conditions and electrotransferred onto PVDF membranes which were probed overnight at 4 °C or for 2 h at room temperature with the primary antibodies listed in Table 1 and then with anti-rabbit (dilution 1:20000; NIF1317, lot9465473, GE Healthcare, Chalfont St. Giles, UK) or anti-mouse (dilution 1:20000; NIF1316, lot6963606, GE Healthcare, Chalfont St. Giles, UK) alkaline phosphatase-conjugated secondary antibodies. Mouse monoclonal anti-β-tubulin I and rabbit polyclonal anti-lamin B1 were used as loading controls of total and cytoplasmic extracts and of nuclear extracts, respectively. Immune complexes were detected with Enhanced ChemiFluorescence reagent (GE Healthcare) in the imaging system Thyphoon^TM FLA 9000 (GE Healthcare). Image analysis was performed with TotalLab TL120 software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).

Western blot was performed as described previously [13 (link)]. Briefly, total (25 µg), cytoplasmic (25 µg) or nuclear (30 µg) proteins were separated by SDS-PAGE under reducing conditions. A commercial mixture of 12 purified pre-stained proteins (NZYColour Protein Marker II, NZYTech, Lisbon, Portugal) was run in each gel to allow for confirmation of the apparent molecular weight of the proteins of interest. The proteins were then electrotransferred onto PVDF membranes (Immobilon^®—P, Merck Millipore Ltd.) which were probed overnight at 4 °C or for 2 h at room temperature with the primary antibodies indicated in Table 1 and then with anti-rabbit (dilution 1:20,000; NIF1317, lot9465473, GE Healthcare, Chalfont St. Giles, UK) or anti-mouse (dilution 1:20,000; NIF1316, lot6963606, GE Healthcare, Chalfont St. Giles, UK) alkaline phosphatase-conjugated secondary antibodies. Mouse monoclonal anti-β-Tubulin I and rabbit polyclonal anti-Lamin B1 were used as a loading controls of total and cytoplasmic extracts and of nuclear extracts, respectively. Immune complexes were detected with Enhanced ChemiFluorescence reagent (GE Healthcare) in the imaging system Thyphoon^TM FLA 9000 (GE Healthcare). Image analysis was performed with TotalLab TL120 software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).