Anti ubxn1 rabbit polyclonal antibody

The proteins in the cell lysate and the eluted product were separated with SDS-PAGE and detected with western blotting. After the proteins were transferred to PVDF membrane, the membrane was cut according to the bands of proteins marker (Precision Plus Protein™ Dual Color Standards, BIO-RAD) to probe multiple proteins. As the primary antibodies, we used anti-myc rabbit polyclonal antibody (Sigma), anti-HA mouse monoclonal antibody (Sigma), anti-HA goat polyclonal antibody (Novus Biologicals), anti-FLAG mouse monoclonal antibody (Sigma), anti-UBXN1 rabbit polyclonal antibody (Millipore), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (Millipore), anti-GST mouse monoclonal antibody (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibody (described in the “immunoprecipitation assay” section), anti-EGFP mouse monoclonal antibody (Clontech), and anti-NiV V rabbit polyclonal antibody, which has been described previously^{51 (link)}. As the secondary antibodies, horseradish-peroxidase-conjugated goat anti-mouse IgG antibody (Dako), anti-rabbit IgG antibody (Dako), or anti-goat IgG antibody (Dako) were used. Chemiluminescence was detected with ECL Prime Western blotting Detection Reagent (GE Healthcare) and an ImageQuant LAS 4000 biomolecular imager (GE Healthcare).

At 24 h posttransfection, the cells were fixed with 4% paraformaldehyde for 30 min. After the cells were washed three times with PBS, they were incubated in blocking buffer (3% bovine serum albumin, 0.1% Triton X-100 in PBS) at room temperature for 30 min. For the staining of myc-tagged NiV V, UBXN1 and HA-tagged UBXN1, anti-myc rabbit polyclonal antibody (Sigma), anti-myc mouse monoclonal antibody (Clontech), anti-UBXN1 rabbit polyclonal antibody (Millipore) and anti-HA mouse monoclonal antibody (Sigma) were incubated with the cell in blocking buffer at 4 °C for over night. For control staining, rabbit serum was used. After the cells were washed three times with wash buffer (0.05% Tween 20 in PBS), they were incubated with Alexa-Fluor-488-conjugated goat anti-rabbit antibody (Invitrogen), Alexa-Fluor-568-conjugated goat anti-mouse antibody (Invitrogen), and Hoechst 33342 (Cambrex) in blocking buffer at room temperature for 1 h. After the cells were washed three times, their immunofluorescence was observed with an IX70 laser confocal microscope and the FluoView FV1000 system (Olympus).