Deltavision spectris microscope

The cells were plated on cover glass (thickness No 1.5) for 24 hr, followed by Flag-ASNA1 plasmid transfection or Flag-FAF1 tetracycline induction for 24 hr, and treated with MG132 for 2 hr where indicated. Cells were then fixed in 3.7% paraformaldehyde/PBS at room temperature for 15 min, permeabilized in 0.2% Triton X-100/PBS for 5 min, and blocked in 3% FBS in PBS/0.05% Tween for 45 min. Antibodies diluted in 3% FBS in PBS/0.05% Tween were sequentially added to the cells and incubated for 1 hr at room temperature followed by three washes with PBS/0.05% Tween. Cells were then incubated for 5 min with 4',6-diamidino-2-phenylindole (DAPI). Finally, the samples were washed three times with PBS/0.05% Tween, three times with PBS, and twice with water before mounting on microscope slides with Mowiol 4-88 (Polysciences, Eppelheim, Germany). Images were obtained with a DeltaVision Spectris microscope (Applied Precision, Issaquah, USA), using a CoolSNAP HQ camera (Roper Scientific, Martinsried, Germany) and a 100× 1.4 NA objective (Olympus, Southend-on-Sea, UK). The SoftWorx software (Applied Precision) was used for image acquisition and deconvolution.

Western analysis was carried out as described previously [13] (link). Production of anti-SUMO and anti-eIF4GI (against the KRERK epitope) antisera has been described elsewhere [41] (link), [42] (link), anti-myc antibodies for immunofluorescence were purified from cell supernatant (cell line CRL1729, from ATCC) using protein G-sepharose or were from Santa Cruz (sc-40), anti-HA antisera were from Santa Cruz (sc-7392) and monoclonal anti-tubulin antibodies were from Sigma (T5168). Immunofluorescence was undertaken as described in Moreno et al. [43] (link). Cells were observed using an Applied Precision Deltavision Spectris microscope using deconvolution software.