Alexa 647 conjugated anti mouse secondary antibody

All antibodies were used at 1 μg/mL for both Western Blot and immunofluorescence. Atto-647N-labelled anti-α K40 acetylated tubulin (C3B9, HPA Cultures) antibody was prepared as previously described.⁵ (link) Mouse monoclonal antibody HPC4 (against the PC tag) was from Roche. Rabbit polyclonal antibody against Par3 was from Merck (#07-330). Mouse monoclonal antibody against aPKC was from Santa Cruz (sc-17837). Rabbit polyclonal anti-GST antibody was from Abcam (#ab19256). Rat monoclonal anti-HA antibody (clone 3F10) was from Roche. Rabbit polyclonal anti-Camsap2 was purchased from Proteinbiotech (17880-1-AP), and rabbit polyclonal anti-Kif2A was purchased from Novus Biologicals (NB500-180). Alexa-555 and Alexa-A647-conjugated anti-rabbit secondary antibodies used were purchased from Thermo (A32732, A21246). Alexa-647-conjugated anti-mouse secondary antibody was also purchased from Thermo (A-11018). Donkey Cy3-conjugated anti-rat antibodies were purchased from Jackson ImmunoResearch.

For analysis of the surface expression of tetherin, 293T cells were cotransfected with tetherin plasmid and plasmid encoding viral antagonist or empty plasmid as negative control. At 48 h post transfection, the cells were washed and harvested in PBS. Expression of tetherin at the cell surface was detected by employing a tetherin-specific mouse monoclonal antibody (BioLegend) at a dilution of 1:50 and an Alexa 647-conjugated anti-mouse secondary antibody at a dilution of 1:100 (ThermoFisher Scientific, Dreieich). Subsequently, cells were fixed with 2% paraformaldehyde and staining was analyzed employing a LSR II Flow Cytometer (BD Biosciences, Heidelberg) and the FACS Diva software (BD Biosiences, Heidelberg). The data were further analyzed using the FCS Express 4 Flow research software (De Novo software).