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
Fig 2: TDP-43 pathology distribution in different regions in TDP-43 PFFs-injected mice. Representative photomicrographs of p409-410 IHC staining in coronal brain sections of cortex (a–d), internal capsule (IC, e–h), midbrain (i–k), medulla oblongata (l–n), The seventh cervical spinal cord (C7, o–q), The third lumber spinal cord (L3, r–t) from TDP-43 M1-C mice analyzed at 8 mpi (n = 3 mice/group) in the ipsilateral (Ipsi) and contralateral (Contra) side of injection. In the cortex, p409–410 staining was mainly detected in the fifth layer of the ipsilateral primary motor cortex (M1, a, c) and contralateral M1 (b, d). e–h Show p409–410 staining in the Ipsi IC (e, g) and Contra IC (f, h). In the midbrain, i–k show p409-410 staining in the Ipsila red nucleus (RN, j), Contra RN (k) and bilateral substantia nigra (SN). In the medulla oblongata, l–n show p409–410 staining in the Ipsi decussatio pyramidum (py, m), Contra py (n) and inferior olive nucleus (ION). o–q show p409–410 staining in the Ipsi anterior horn of C7 (p) and Contra anterior horn of C7 (q). r–t show p409–410 staining in the Ipsila anterior horn of L3 (s) and Contra anterior horn of L3 (t). [Scale bars, a–b, 200 µm; c–d, p–q, s–t, j–k 50 µm; e–f, 100 µm; g–h, m–n, 20 µm; i, l, o, r, 500 µm]
Fig 3: Heat maps of TDP-43 pathology in coronal sections stained with pTDP-43 antibody. Semi-quantitative analyses of the burden of TDP-43 pathology in neurons and in white matter tracts in the brain and spinal cord of TDP-43 M1-C mice at different times (n = 3 at 1 mpi, n = 3 at 2 mpi, n = 3 at 6 mpi, n = 3 at 12 mpi) after TDP-43 PFFs injection. Each panel represents heat map pathology distribution on one of the six different coronal planes (Bregma 0.74 mm, - 1.70 mm, - 3.80 mm, - 7.64 mm, C7 section, L3 section) for the different post-injection times. The mean values of the TDP-43 pathology were graded from negative (0, gray) to most abundant (3, red) and color-coded onto heat maps. The panels in the far left column shows sagittal views of the corresponding coronal planes (blue line) and the site of injection into cortex was indicated by the red star.
Fig 4: Double immunofluorescence analysis and Western blot analysis of TDP-43 PFFs M1-C mice at 4 mpi. a1–e3 Representative images of immunofluorescence were analyzed to demonstrate the aggregation of pathological TDP-43 in different types of cells. Double immunofluorescence analysis of cortex, pons, medulla oblongata and cervical spinal cord from TDP-43 PFFs M1-C mice for pTDP-43 (red, a2-e2) with MAP2 in M1(green, a1) and parvicellular reticular nucleus (green, b1), ubiquitin in cervical spinal cord (green, c1), GFAP in LGP (green, d1), Iba-1 in MdD (green, e1). f1–g3 Double immunolabeling for MBP (green) and neurofilament (red) in dorsal corticospinal tract in the ipsilateral side (f1–f3) and contralateral side (g1–g3) of injection from TDP-43 PFFs M1-C mice. Co-immunolabeling is represented by signal in yellow. Cell nuclei were counterstained with Hoechst 33258 (blue). [Scale bars, 30 µm (a1–g3)]. WB analysis of pTDP-43 in the soluble and insoluble fractions from cortex (h), red nucleus (j), decussatio pyramidum (l) and cervical spinal cord anterior horn (n) using the anti-pTDP-43 (phosphorylated at Ser409/Ser410) antibody. Blots were probed for GAPDH as a loading control (Bottom). Molecular weight markers of migrated protein standards are expressed in KDa. Quantification of soluble and insoluble pTDP-43 levels in cortex (i), red nucleus (k), decussatio pyramidum (m) and cervical spinal cord anterior horn (o) (n = 3 mice/group) show plentiful pTDP-43 in the extracts of TDP-43 PFFs M1-C mice while few in age-matched PBS M1-C mice. The error bar in panels (i, k, m, o) represents the Standard Error of Mean (SEM). Data are the mean ± SEM. Statistical significance was analyzed using the Student’s t test and Mann–Whitney test, ***P < 0.001.
Fig 5: ITSN1 SH3 domains mediate direct binding to SAM68 in cellular context and in vitro. a Schematic representation of the domain structures of ITSN1s truncated forms used in the microtubule bench assay analysis to reveal the interaction between ITSN1s and SAM68. b Western blot analysis of the total lysates of HEK cells transfected with plasmids encoding indicated GFP-fused truncated forms of ITSN1s. The proteins were detected with anti-GFP antibodies. c The results of the microtubule bench assay confirming the interaction between SH3-containing truncated forms of ITSN1s and SAM68. HeLa cells were co-transfected with the construction encoding SAM68 fused to RFP-MBD and the plasmid expressing one of the truncated forms of ITSN1s fused to GFP. The line profile representing the fluorescence intensity from two channels is shown next to the respective micrograph. Scale bar: 15 µm. d Scatter plot representing the co-localization level between RFP-MBD-fused SAM68 or RFP-MBD alone (Supplementary Figure S6A) and truncated forms of ITSN1s. 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 < 0.05, ***p < 0.0005, n.s. not significant, two-tailed t test. e Pull-down assay confirming the direct binding of ITSNSH3 to SAM68 in vitro. TDP43 was used as a negative control, whereas SH3 domain of SRC kinase (SRCSH3) known to interact with SAM68 was used as a positive control. Proteins were visualized using Coomassie staining. Red arrows indicate GST-SRCSH3 and GST-ITSN1SH3 precipitated by SAM68-His. White asterisks indicate nonspecific products obtained during SAM68 purification
Supplier Page from Abcam for Recombinant Human TDP43 protein (denatured)