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. 2019 Apr 16;14(4):e0215062. doi: 10.1371/journal.pone.0215062

Fig 3. Subcellular colocalization and co-immunoprecipitation of the intrabody and IFNα4 upon transient transfection of HEK293T cells.

Fig 3

Immunofluorescence analysis by laser scanning confocal microscopy of fixed and permeabilized HEK293T cells transiently co-transfected with the myc-tagged αIFNα-ib and the Twin-Strep-tagged IFNα4 expression plasmids (A) or myc-tagged control intrabody (αTLR2-ib, B). Expression of IFNα4 and αIFNα-ib was analysed with StrepMAB Classic Chromeo 546 antibody and goat anti-c-myc-FITC antibody, respectively. Expression of calnexin was visualized with rabbit anti-calnexin and goat anti-rabbit Cy5 antibodies. C, As a negative control, cells were co-transfected with the IFNα4 expression plasmid and the empty intrabody vector pCMV/myc/ER. Staining as in A resulted in no signal. D, Co-immunoprecipitation. HEK293T cells were co-transfected with the αIFNα-ib and IFNα4 plasmids. Negative controls were transfected with IFNα4 and the unrelated, myc-tagged intrabodies αTLR2-ib or αNCAM-ib or the empty vector pCMV/myc/ER. 48 h after transfection, co-immunoprecipitation was performed with anti-myc agarose beads, and eluted proteins were visualized by immunoblotting using an anti-c-myc antibody for detection of the intrabodies or an anti-StrepTag antibody for detection of IFNα4.