Skip to main content
. 2020 Nov 13;10:559915. doi: 10.3389/fcimb.2020.559915

Figure 7.

Figure 7

AnkF binds the type III intermediate filament vimentin. (A) A yeast-two-hybrid assay was performed with the Matchmaker Gold Yeast Two-Hybrid System (Clontech) and a HeLa cDNA library. Recombinant, leucine-tryptophan auxotrophic yeast Y190 were grown on SCAD agar plates in the absence (1, 2, 5 and 6) and presence of leucine and tryptophan and X-Gal (3, 4, 7 and 8). (1 and 3) Recombinant yeast carrying the empty vector pGADH with the GAL4 activation domain and the vector pGBT encoding ankF fused to the GAL4 binding domain. (2 and 4) Recombinant Y190 carrying the empty vector pGBT and the vector pGADH-vimentin. (5 and 7) Recombinant Y190 carrying the vector p53pBD and vector pSV40-pAD-gal4 containing the large T-antigen of the SV40 virus fused to the Gal4 activation domain as positive control interaction partners. (6 and 8) Recombinant Y190 carrying the vector pGBT9-ankF and the vector pGADH-vimentin. The image is representative of three independent experiments. (B, C) Stably-transfected HeLa-AnkF cells (HeLa-pWHE644/655-AnkF), harboring a doxycycline-inducible AnkF-expression system, were incubated without or with 1 µg/ml doxycycline for the indicated durations. (B) Cell lysates were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with antibodies against AnkF, vimentin and actin as loading control. A representative image of three independent experiments is shown. (C) Total isolated RNA was reverse-transcribed using SuperScript II reverse transcriptase according to the manufacturer’s protocol and a qRT-PCR was performed with primers specific for vimentin (936 and 937) and actin (827 and 828) as a housekeeping gene. Vimentin was normalized to actin and is depicted as fold change compared to non-induced cells. (D) CHO-FcR cells were transiently transfected with plasmids pCMV-HA-vimentin (red) together with pGFP (green) alone (upper panel) or together with pEGFP-AnkF (lower panel). 24 h post-transfection, cells were fixed and HA-vimentin was stained with specific antibodies by indirect immunofluorescence (red). Nuclei were stained with DAPI (blue). Transient expression of HA- and GFP-containing proteins was visualized by epifluorescence microscopy. Images are representative of three independent experiments.