Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Analysis of CAPN3 binding to the two titin regions in cells with heterologous overexpression. A , schematic illustration of the mCherry-fusion proteins with the two regions of human titin (NCBI reference sequence: NP_596869.4 ). The N2A region corresponds to the mouse mCN48 fragment identified as a p94-binding fragment via yeast two-hybrid screening in our previous study . B , EGFP-CAPN3:CS is coimmunoprecipitated with mCherry-titin CT more efficiently than with mCherry-titin I80-PEVK in HEK293T cells. Because of their weak interaction, the cells were treated with a dithiobis(succinimidyl propionate) cross-linker before cell lysis and immunoprecipitation. C , band intensity ratio of the coimmunoprecipitated CAPN3 to the CAPN3 in the lysate. ∗ p = 0.0211, Mann–Whitney U test . D , immunostaining of EGFP-CAPN3:CS ( green ) coexpressed with mCherry fusion proteins of the two titin regions ( magenta ) in HeLa cells. Arrows ( upper panel ) and arrowheads ( bottom panel ) show filopodia with no GFP staining. The scale bar represents 20 μm. E , translocation of EGFP-CAPN3:CS to the membrane fractions by mCherry-titin CT-kRas, but not mCherry-titin I80-PEVK-kRas, in HeLa cells. HeLa cells were fractionated into cytosol and membrane fractions. The upper and the lower panels show the expression of EGFP-CAPN3:CS (Blot: anti-CAPN3 antibody), and mCherry-titin-kRas (Blot: anti-RFP antibody), respectively, and the protein bands were labeled with arrows . F , ratio of band intensity of EGFP-CAPN3:CS in the membrane fraction to that in the cytosol fraction. ∗∗ p = 0.0073, one-way ANOVA with Dunnett’s multiple comparison test. a.a., amino acid number; LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain; EGFP, enhanced green fluorescent protein; CT, COOH terminal; RFP, red fluorescent protein.

Article Snippet: The antibodies used in this study included rabbit anti-CAPN3 polyclonal antibody (Proteintech, Tokyo, Japan, 28476-1-AP), anti-α-actinin (Sigma-Aldrich, EA-53), anti-GFP polyclonal antibody (MBL, Tokyo, Japan, Code No. 598), and anti-red fluorescent protein monoclonal antibody cocktail (MBL, Code No. M208–3), and anti-mCherry antibody (Proteintech, 26765-1-AP).

Techniques: Binding Assay, Over Expression, Sequencing, Two Hybrid Screening, Lysis, Immunoprecipitation, MANN-WHITNEY, Immunostaining, Staining, Translocation Assay, Membrane, Expressing, Labeling, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Localization of LGMDR1 mutants of EGFP-CAPN3 and mCherry-titin CT in HeLa cells. A , immunostaining of EGFP-CAPN3:CS ( green ) and its LGMDR1 mutants coexpressed with mCherry-titin CT ( magenta ) in HeLa cells. The scale bar represents 10 μm. B , quantification of the colocalization of EGFP-CAPN3:CS and its LGMDR1 mutants with mCherry-titin CT. Pearson correlation coefficient was calculated using Coloc2 software. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain; EGFP, enhanced green fluorescent protein; CT, COOH terminal.

Article Snippet: The antibodies used in this study included rabbit anti-CAPN3 polyclonal antibody (Proteintech, Tokyo, Japan, 28476-1-AP), anti-α-actinin (Sigma-Aldrich, EA-53), anti-GFP polyclonal antibody (MBL, Tokyo, Japan, Code No. 598), and anti-red fluorescent protein monoclonal antibody cocktail (MBL, Code No. M208–3), and anti-mCherry antibody (Proteintech, 26765-1-AP).

Techniques: Immunostaining, Software, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Partial rescues of abnormal intracellular localization of EGFP-CAPN3:CS:A702V but not of EGFP-CAPN3:CS:D705H in Capn3 +/+ mice. A , immunostaining of actinin ( magenta ) and EGFP-CAPN3:CS:A702V and EGFP-CAPN3:CS:D705H ( green ) in the TA muscles of Capn3 +/+ mice. Right panel shows the line plot of the merged image. The scale bar represents 10 μm. B , coexpression of CAPN3:CS ameliorated the localization of EGFP-CAPN3:CS:A702V but not EGFP-CAPN3:CS:D705H at the mCherry-titin CT ( magenta ) enriched plasma membrane in HeLa cells. Right panels show the line plot of mCherry-titin CT ( magenta ) and EGFP-CAPN3:CS, CAPN3:CS:A702V, and CAPN3:CS:D705H ( green ) within the left merged images. C , quantification of the colocalization of EGFP-CAPN3:CS and its LGMDR1 mutants with mCherry-titin CT upon coexpression of CAPN3 in HeLa cells. Pearson correlation coefficient was calculated using Coloc2 software. ∗∗∗ p = 0.0019, one-way ANOVA with Bonferroni multiple comparison test. LGMDR1, limb-girdle muscular dystrophy R1; CT, COOH terminal; CAPN, calpain; EGFP, enhanced green fluorescent protein; TA, tibialis anterior.

Article Snippet: The antibodies used in this study included rabbit anti-CAPN3 polyclonal antibody (Proteintech, Tokyo, Japan, 28476-1-AP), anti-α-actinin (Sigma-Aldrich, EA-53), anti-GFP polyclonal antibody (MBL, Tokyo, Japan, Code No. 598), and anti-red fluorescent protein monoclonal antibody cocktail (MBL, Code No. M208–3), and anti-mCherry antibody (Proteintech, 26765-1-AP).

Techniques: Immunostaining, Muscles, Clinical Proteomics, Membrane, Software, Comparison