Fig. 1.
Scaffold protein ITSN1 localizes in HeLa cells nuclei and interacts with RBP SAM68 in the microtubule bench assay. a, left panel. Cellular distribution of endogenous ITSN1 in HeLa cells. ITSN1 was detected using anti-ITSN1 antibodies and Alexa Fluor 594-conjugated secondary antibodies. Nucleus was visualized using DAPI staining. Scale bar: 15 µm. a, right panel. Cellular distribution of the overexpressed ITSN1 short isoform (ITSN1s) in HeLa cells. HeLa cells were transfected with the plasmid encoding GFP-fused ITSN1s. Cells were fixed 24 h post-transfection. Nucleus was visualized using DAPI staining. Scale bar: 15 µm. b The results of the microtubule bench assay performed to identify the interaction between ITSN1 and selected RBPs. HeLa cells were co-transfected with the construction encoding ITSN1 fragment containing SH3 domains (ITSN1SH3) fused to RFP-MBD and the plasmid expressing one of the four tested RBPs (SAM68, WBP11, LARP6 or hnRNPK) fused to GFP. Scale bar: 15 µm. The line profile representing the fluorescence intensity from two channels is shown next to the respective microphotograph. c Scatter plot representing the co-localization level of MBD-fused ITSN1SH3 with one of the four tested RBPs and MBD with SAM68 as a control. Each data point represents a correlation coefficient between fluorescence intensities from red and green channels along the line crossing microtubules. The plot shows the data from three independent experiments. Lines show mean values. ***p < 0.0005, two-tailed t test. d HeLa cells were co-transfected with the constructions encoding SAM68 (upper panel) or TDP43 (lower panel) fused to RFP-MBD and the plasmid expressing full-length ITSN1s fused to GFP. TDP43 was used as a negative control as it binds RNA but it lacks potential ITSN1-interacting motifs. The line profile representing the fluorescence intensity from two channels is shown next to the respective micrograph. Scale bar: 15 µm. The scatter plot represents the co-localization level of MBD-fused SAM68 or TDP-43 with the full-length ITSN1s. Each data point represents a correlation coefficient between fluorescence intensities from red and green channels along a line crossing microtubules. The plot shows the data from three independent experiments. Lines show mean values. ***p < 0.0005, two-tailed t test