Anti hnrnp q

SH-SY5Y cells (3 × 10⁵) and NSC-34 (3.5 × 10⁵) were plated in 6-well plates containing coverslips. For SH-SY5Y treated with siRNA against hnRNPs and NSC-34 we plated the corresponding number of cells in 6-well plates containing coverslips coated with poly-L-lysine solution at a final concentration of 0.01% (w/v) in H20 (SigmaAldrich, St Louis, MO, USA). After 24 h, cells were washed three times with PBS, fixed in 3.2% paraformaldehyde in PBS for 1 h at room temperature and permeabilized by using 0.3% Triton in PBS for 5 min on ice. Cells were then blocked with 2% BSA/PBS for 20 min at room temperature and immunolabeled with 1:200 rabbit polyclonal antibody anti-hnRNP Q (SigmaAldrich, St Louis, MO, USA) or 1:200 rabbit polyclonal antibody anti-hnRNP R (Abcam, Cambridge, UK) in 2% BSA/PBS overnight at 4°C. Next day, cells were washed three times with PBS, incubated with 1:500 anti-rabbit Alexa-Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature and coverslipped with Vectashield-DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA). Each slide was analyzed at the microscopy facility of University of Trieste, using a Nikon Eclipse C1si confocal microscope system mounted on a Nikon TE-2000U inverted microscope with a 60X objective.

For immunoblotting, cells were disrupted with complete protein solubilizing buffer containing 1% SDS and 2M urea in PBS, followed by sonication. Immunoblot analyses were performed with polyclonal anti-CRY1, monoclonal anti-GAPDH (Millipore), polyclonal anti-14-3-3ζ (Santa Cruz Biotechnology), and polyclonal anti-hnRNP Q (Sigma-Aldrich) antibodies. Horseradish peroxidase-conjugated mouse (Thermo Scientific) and rabbit (Jackson ImmunoResearch Laboratories) secondary antibodies were visualized with SUPEX ECL reagent (Neuronex) and a LAS-4000 system (FUJI FILM), according to the manufacturer’s instructions. Acquired images were further analyzed with the Image Gauge program (FUJIFILM).