recombinant human importin beta  (Novus Biologicals)

Journal: Neurotherapeutics

Article Title: Small molecule JRMS modulating importin-β1 chaperone activity as a therapeutic strategy reducing TDP-43 pathology

doi: 10.1016/j.neurot.2026.e00834

Figure Lengend Snippet: JRMS reduces TDP-25 aggregates in mouse primary cortical neurons. (a) Neurons treated with up to 30 μM JRMS exhibit no significant cytotoxicity. (b) Immunofluorescence imaging showing reduction of TDP-25 aggregates (green; arrows) in JRMS-treated neurons compared to DMSO control. DAPI (blue) Quantification showing reduced (c) number and (d ) size of aggregates in JRMS-treated neurons compared to DMSO control. (e) Immunoblot of total lysates in neurons transduced with EGFP, EGFP-TDP-25 and EGFP-TDP-43, treated with 15 μM JRMS or DMSO, labelled for KPNB1, EGFP (JL8 antibody), pTDP (409/410) and GAPDH. JRMS reduces phosphorylated TDP-25 (arrow) without affecting the total level of expression of the EGFP-tagged proteins. Quantification shows similar levels of EGFP-TDP-25 (f) , and reduced phosphorylated TDP-25 (g) in JRMS-treated neurons compared to DMSO control. (h) Quantification of KPNB1 levels shows no difference between JRMS-treated and DMSO control. Scale bar = 20 μm; ∼250 neurons across N = 3 biological replicates per treatment; Statistical analysis was performed using two-tailed t -test ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001 .

Article Snippet: Membranes were then incubated overnight at 4 °C with the following primary antibodies diluted in blocking solution: anti-phospho-TDP-43 (pS409/410; mouse, 1:1000; CosmoBio, #TIP-PTD-M01), anti- KPNB1 (mouse, 1:1000; Santa Cruz Biotechnology, #SC137016), anti-GFP (mouse, 1:5000; Clontech, #632380), and anti-GAPDH (mouse, 1:10,000; Abcam, #ab8245).

Techniques: Immunofluorescence, Imaging, Control, Western Blot, Transduction, Expressing, Two Tailed Test

Journal: Neurotherapeutics

Article Title: Small molecule JRMS modulating importin-β1 chaperone activity as a therapeutic strategy reducing TDP-43 pathology

doi: 10.1016/j.neurot.2026.e00834

Figure Lengend Snippet: The effect of JRMS on reducing TDP-25 aggregation is dependent on KPNB1. (a) Immunoblot showing that siRNA knockdown of KPNB1 increases insoluble and phosphorylated EGFP-TDP-25 (pTDP), whereas increased expression of KPNB1-EGFP reduces insoluble and phosphorylated Flag-TDP-25. (b) Quantification of amount of insoluble pTDP, normalized to GAPDH within each fraction. (c) Immunofluorescence image showing expression of KPNB1-EGFP (green) reduces Flag-TDP-25 aggregates (red; arrowhead), quantified in (d) . DAPI (blue). (e) Immunoblot showing effect of JRMS in reducing insoluble, phosphorylated EGFP-TDP-25 is prevented in cells with siRNA knockdown of KPNB1. (f) The quantification of insoluble pTDP faction in DMSO vs JRMS in siScram and siKpnB1 cells. (g) JRMS treatment increases the cytoplasmic localization of KPNB1-EGFP, quantified in (h) . (i) Immunoblot showing that endogenous KPNB1 co-immunoprecipitates with EGFP-TDP-25, with apparent reduced association in conditions of JRMS treatment as quantified in (j) . However, (k) normalization to pTDP shows increased interaction, indicating that JRMS promotes KPNB1 binding to aggregated TDP-25. (l) JRMS treatment does not affect nucleocytoplasmic ratio of endogenous TDP-43 (red), quantified in ( m ) or of the ( n ) NLS-tdTomato-NES NCT reporter (red), quantified in (o) . DAPI (blue). Scale Bar = 10 μm; N = 3 biological replicates; ∼100 cells quantified per biological replicate in C, G, L and N; Statistical analysis was performed using two-tailed t -test for two condition comparison or one-way ANOVA for multi-condition comparison ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001 .

Article Snippet: Membranes were then incubated overnight at 4 °C with the following primary antibodies diluted in blocking solution: anti-phospho-TDP-43 (pS409/410; mouse, 1:1000; CosmoBio, #TIP-PTD-M01), anti- KPNB1 (mouse, 1:1000; Santa Cruz Biotechnology, #SC137016), anti-GFP (mouse, 1:5000; Clontech, #632380), and anti-GAPDH (mouse, 1:10,000; Abcam, #ab8245).

Techniques: Western Blot, Knockdown, Expressing, Immunofluorescence, Binding Assay, Two Tailed Test, Comparison

Journal: Neurotherapeutics

Article Title: Small molecule JRMS modulating importin-β1 chaperone activity as a therapeutic strategy reducing TDP-43 pathology

doi: 10.1016/j.neurot.2026.e00834

Figure Lengend Snippet: JRMS prevents/reverses TDP-25 aggregation in vivo . (a) Immunohistochemistry of DMSO or JRMS treated AAV9-EGFP-TDP-25 (green) mouse brains labelled with neuronal marker (NeuN; magenta) and phosphorylated TDP-43 S409/410 (pTDP; red; arrowhead). DAPI (blue). There is a lower abundance of phosphorylated TDP-25 aggregates (arrows) with JRMS treatment compared to DMSO control. Scale Bar = 55 μm. TDP-25 aggregates are smaller in JRMS-treated mice as quantified in (b) and show a trend toward decreased abundance as quantified in (c) . (d) Immunoblot of DMSO and JRMS-treated mice showing equivalent expression levels of KPNB1 and EGFP-TDP-25, but a ∼30 % decrease in levels of phosphorylated TDP-25, an indicator of reduced aggregation, as quantified in (e) . (f) There is no detectable change in neuronal density in DMSO and JRMS-treated mice, supporting that there is no toxicity associated with JRMS. (g) JRMS treatment affects KPNB1 (red) subcellular localization (NeuN in magenta; DAPI in Blue), increasing its cytoplasmic concentration (Scale Bar = 10 μm), quantified in (h ; ∼50 neurons per mouse ) . N = 4 mice per treatment condition for immunohistochemical analysis and N = 8 mice per treatment condition for biochemical analysis. Statistical analysis was performed using two-tailed t -test ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001 .

Article Snippet: Membranes were then incubated overnight at 4 °C with the following primary antibodies diluted in blocking solution: anti-phospho-TDP-43 (pS409/410; mouse, 1:1000; CosmoBio, #TIP-PTD-M01), anti- KPNB1 (mouse, 1:1000; Santa Cruz Biotechnology, #SC137016), anti-GFP (mouse, 1:5000; Clontech, #632380), and anti-GAPDH (mouse, 1:10,000; Abcam, #ab8245).

Techniques: In Vivo, Immunohistochemistry, Marker, Control, Western Blot, Expressing, Concentration Assay, Immunohistochemical staining, Two Tailed Test

Journal: Neurotherapeutics

Article Title: Small molecule JRMS modulating importin-β1 chaperone activity as a therapeutic strategy reducing TDP-43 pathology

doi: 10.1016/j.neurot.2026.e00834

Figure Lengend Snippet: JRMS reduces TDP-25 aggregates in mouse primary cortical neurons. (a) Neurons treated with up to 30 μM JRMS exhibit no significant cytotoxicity. (b) Immunofluorescence imaging showing reduction of TDP-25 aggregates (green; arrows) in JRMS-treated neurons compared to DMSO control. DAPI (blue) Quantification showing reduced (c) number and (d ) size of aggregates in JRMS-treated neurons compared to DMSO control. (e) Immunoblot of total lysates in neurons transduced with EGFP, EGFP-TDP-25 and EGFP-TDP-43, treated with 15 μM JRMS or DMSO, labelled for KPNB1, EGFP (JL8 antibody), pTDP (409/410) and GAPDH. JRMS reduces phosphorylated TDP-25 (arrow) without affecting the total level of expression of the EGFP-tagged proteins. Quantification shows similar levels of EGFP-TDP-25 (f) , and reduced phosphorylated TDP-25 (g) in JRMS-treated neurons compared to DMSO control. (h) Quantification of KPNB1 levels shows no difference between JRMS-treated and DMSO control. Scale bar = 20 μm; ∼250 neurons across N = 3 biological replicates per treatment; Statistical analysis was performed using two-tailed t -test ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001 .

Article Snippet: The pcDNA3.1 constructs for transfection have been described previously, and include: EGFP, EGFP-TDP-25, EGFP-TDP-35, EGFP-TDP-43ΔNLS (K82A, R83A and K84A), EGFP-TDP-C-spl-272, RFP-TDP-25, 3 × FLAG-mTB-TDP-25 [ , ] and KPNB1-EGFP (Addgene plasmid # 106941).

Techniques: Immunofluorescence, Imaging, Control, Western Blot, Transduction, Expressing, Two Tailed Test

Journal: Neurotherapeutics

Article Title: Small molecule JRMS modulating importin-β1 chaperone activity as a therapeutic strategy reducing TDP-43 pathology

doi: 10.1016/j.neurot.2026.e00834

Figure Lengend Snippet: The effect of JRMS on reducing TDP-25 aggregation is dependent on KPNB1. (a) Immunoblot showing that siRNA knockdown of KPNB1 increases insoluble and phosphorylated EGFP-TDP-25 (pTDP), whereas increased expression of KPNB1-EGFP reduces insoluble and phosphorylated Flag-TDP-25. (b) Quantification of amount of insoluble pTDP, normalized to GAPDH within each fraction. (c) Immunofluorescence image showing expression of KPNB1-EGFP (green) reduces Flag-TDP-25 aggregates (red; arrowhead), quantified in (d) . DAPI (blue). (e) Immunoblot showing effect of JRMS in reducing insoluble, phosphorylated EGFP-TDP-25 is prevented in cells with siRNA knockdown of KPNB1. (f) The quantification of insoluble pTDP faction in DMSO vs JRMS in siScram and siKpnB1 cells. (g) JRMS treatment increases the cytoplasmic localization of KPNB1-EGFP, quantified in (h) . (i) Immunoblot showing that endogenous KPNB1 co-immunoprecipitates with EGFP-TDP-25, with apparent reduced association in conditions of JRMS treatment as quantified in (j) . However, (k) normalization to pTDP shows increased interaction, indicating that JRMS promotes KPNB1 binding to aggregated TDP-25. (l) JRMS treatment does not affect nucleocytoplasmic ratio of endogenous TDP-43 (red), quantified in ( m ) or of the ( n ) NLS-tdTomato-NES NCT reporter (red), quantified in (o) . DAPI (blue). Scale Bar = 10 μm; N = 3 biological replicates; ∼100 cells quantified per biological replicate in C, G, L and N; Statistical analysis was performed using two-tailed t -test for two condition comparison or one-way ANOVA for multi-condition comparison ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001 .

Article Snippet: The pcDNA3.1 constructs for transfection have been described previously, and include: EGFP, EGFP-TDP-25, EGFP-TDP-35, EGFP-TDP-43ΔNLS (K82A, R83A and K84A), EGFP-TDP-C-spl-272, RFP-TDP-25, 3 × FLAG-mTB-TDP-25 [ , ] and KPNB1-EGFP (Addgene plasmid # 106941).

Techniques: Western Blot, Knockdown, Expressing, Immunofluorescence, Binding Assay, Two Tailed Test, Comparison