Mouse anti ubiquitin

Mouse anti-Fbxo7 (Santa Cruz, sc-271763), mouse anti-GAPDH (Santa Cruz, sc-365062), mouse anti-Flag (Sigma, F1804), mouse anti-β-Actin (Sigma, A2228), mouse anti-Myc (Santa Cruz, sc-40), mouse anti-V5 (Bio-Rad, MCA1360GA), mouse anti-Ubiquitin (Abcam, ab134953), rabbit anti-Bag2 (Abcam, ab79406), mouse anti-HA (Abcam, ab130275), anti-mouse horseradish peroxidase-conjugated secondary antibody (Rockland, 18-8817-31) and anti-rabbit horseradish peroxidase-conjugated secondary antibody (Rockland, 18-8816-31) for IP Western blots, anti-mouse horse radish peroxidase-conjugated secondary antibodies (Millipore, AP187P) and anti-rabbit horse radish peroxidase-conjugated secondary antibodies (Millipore, 12-348) for regular Western blots. All primary antibodies were used at 1:5000 dilution, except anti- β-Actin, which was diluted 1:50,000 for Western blot analyses. The secondary antibodies for IP were diluted 1:5000. The secondary antibodies for Western blots were diluted 1:10,000.

NRK cells were fixed with 4% PFA, 150 mM sucrose in PBS for 20 min at 37°C. After fixation, cells were then washed and incubated for 12 h at 4°C with either rabbit anti-YB-1, mouse anti-HuR antibody (Molecular Probes, 10 μg/ml), mouse monoclonal anti-tubulin antibody E7 (1:2000 dilution), rabbit anti-phospho-eIF2α antibody (Cell Signaling, 1:2000 dilution), mouse anti-ubiquitin (abcam, ab7254, 1:1000 dilution), goat anti-vimentin (Santa Cruz, SC-7558, 1:1000 dilution) and goat anti-TIA-1 (Santa Cruz, SC-1751, 1:200 dilution). Cells were then washed extensively in PBS and incubated for 1 h with fluorochrome (Alexa Fluor^®488 and 594)-coupled secondary antibodies (Invitrogen) in blocking solution. After final washes with PBS, samples were mounted for fluorescence microscopy analysis. The measurements of the mean GFP, anti-HuR or anti-TIA-1 fluorescence intensities were performed using the ImageJ software. The mean integrated intensity per area unit was measured in the specified locations (nucleus, cytoplasm). Integrated intensities of stress granules were defined as the intensity of the anti-YB-1 immunofluorescence signal integrated over the sum of the granule areas minus the integrated intensity in the surrounding cytoplasmic background.