Alexa fluor 568 conjugated goat anti mouse antibody

Mouse antibody against Flag-tag (M2) and M2-conjugated agarose were purchased from Sigma-Aldrich (St. Louis, MO); rabbit antibodies against TOM20 and the p65 subunit of NF-κB were from Santa Cruz Biotechnology (Dallas, TX); Alexa Fluor 488 conjugated goat anti-mouse and anti-rabbit antibodies, Alexa Fluor 568 conjugated goat anti-mouse antibody, and Alexa Fluor 633 conjugated goat anti-rabbit antibody were from Invitrogen (Carlsbad, CA). Sendai virus (SeV, Cantell strain, Charles River Laboratories) was used at 100 hemagglutination (HA) units/ml culture media. HEK293T, HEK293T-IFNβ-luciferase, Mavs^−/− MEF cells and derivatives were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) cosmic calf serum (Hyclone, Thermo Fisher Scientific, Waltham, MA) with penicillin (100 U/ml) and streptomycin (100 μg/ml). Other chemicals and reagents were from Sigma-Aldrich unless otherwise specified.

In order to test the specificity of CAG-driven eGFP expression during cardiac differentiation, immunostaining of cardiac troponin I (cTnI) was performed in 6 + 24-day-old adherent EBs. Samples were fixed with 4% paraformaldehyde in Dulbecco's modified phosphate buffered saline (D-PBS, Sigma) for 15 min at room temperature, followed by three washing steps with D-PBS. To block nonspecific antibody binding, samples were incubated in D-PBS containing 2 mg/mL bovine serum albumin, 1% fish gelatin, 5% goat serum, and 0.1% Triton-X 100 for 1 h at room temperature. The samples were then incubated with primary antibodies (monoclonal mouse anti-cTnI, Sigma) at the dilution of 1 : 500 for 1 h at room temperature. After washing with D-PBS, the cells were incubated with secondary antibodies (Alexa Fluor 568-conjugated goat anti-mouse antibody, Invitrogen) for 1 h at room temperature. Secondary antibodies were diluted in the blocking solution at 1 : 250. 4′,6-Diamidino-2-phenylindole·2HCl (DAPI, Invitrogen) was used for nuclear staining (10 μM, for 10 min in D-PBS). The stained samples were examined by an Olympus fluorescence microscope.

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).