Mouse monoclonal anti ha 11

Immunofluorescence experiments were performed 48 h post-transfection in HeLa and MCF7 cells as described previously (Smith et al. 2009 (link)). Primary antibodies used included mouse monoclonal anti-HA.11 (1:200; BioLegend, San Diego, CA, USA), rabbit polyclonal anti-HA (Y-11) (Santa Cruz Biotechnology), mouse monoclonal anti-FLAG M2 (1:200; Sigma), rabbit polyclonal anti-PDI (1:200; Cell Signaling Technology), rabbit monoclonal anti-Golgin-97 (1:200; Cell Signaling Technology) and rabbit polyclonal anti-NIS ab104920 (1:500; Abcam). Cells were visualised using a Zeiss Axioplan fluorescent microscope (Zeiss) with a 100× objective.

Cells were harvested in radioimmunoprecipitation assay (RIPA) (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% vol/vol IGEPAL CA-630, 6 mM sodium deoxycholate, 1 mM EDTA) with protease and phosphatase inhibitor cocktails (Sigma). Western blotting was performed as described previously (Smith et al. 2012 (link)). Proteins (30 µg) were separated by SDS-PAGE using a 15% acrylamide gel (12% for the detection of NIS). Primary antibodies used were mouse monoclonal anti-HA.11 (1:1000; BioLegend), mouse monoclonal anti-FLAG M2 (1:500; Sigma), anti-PBF antibody (made by Eurogentec (Seraing, Belgium) using the full-length PBF protein as an epitope), mouse monoclonal anti-MYC-Tag 9B11 (1:1000; Cell Signaling Technology) and mouse monoclonal β-actin AC-15 (1:10,000; Sigma).