capn3 antibody  (Proteintech)

Journal: Scientific Reports

Article Title: Myoblast paracrine factors induce catabolic remodelling in electrically stimulated myotubes

doi: 10.1038/s41598-025-21126-y

Figure Lengend Snippet: EPS-induced structural and molecular alterations to myotubes are influenced by MBL exposure. ( A ) Transmission electron microscopy images of unstimulated (NS) C 2 C 12 myotubes and immediately post-electrical pulse stimulation (EPS). White arrows identify an intact Z line and yellow arrows denote Z line streaming. Scale bar = 1200 nm. ( B ) Cropped representative western blots from the same gel, delineated by a perforated line. ( C ) A graphical summary of β-dystroglycan, desmin, heat shock protein 70 (HSP70), calpain 3, phosphorylated acetyl CoA carboxylase at serine 79 (p-ACC ser79 ), total (t-)ACC, phosphorylated Unc-51-like kinase 1 at serine 555 (p-ULK1 ser555 ), t-ULK1, phosphorylated mammalian target of rapamycin at serine 2448 (p-mTOR ser2448 ), t-mTOR, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 at threonine 37&46 (p-4EBP1 Thr37/46 ), and t-4EBP1 protein content in resting and stimulated myotubes. ( D ) A visual schematic of the study design. Differentiated myotubes and media were collected following 1-hr of electrical pulse stimulation (EPS) or unstimulated incubation in fresh growth media. Transwells with (MBL+) or without (MBL–) myoblasts were inserted into the culture at 0-hrs post-EPS, and cell lysates were collected at various time points thereafter. ( E ) Creatine kinase (CK) activity in conditioned media of EPS damaged MBL + and MBL– myotubes. F - H ) Representative western blots of β-dystroglycan and desmin protein expression and accompanying graphical representations of EPS damaged MBL + and MBL– myotubes. N = 3. Data are expressed as means ± SEM. * p < 0.05 effect of MBL, # p < 0.05 effect of time. Two-tail independent Student’s t-test ( C ) and two-way ANOVA ( E , G , H ). Original western blots are presented in Supplementary Fig. 1.

Article Snippet: Clpn3 , Applied Biosystems , 1,925,544 , Mm00482985_m1.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Binding Assay, Incubation, Activity Assay, Expressing

Journal: Scientific Reports

Article Title: Myoblast paracrine factors induce catabolic remodelling in electrically stimulated myotubes

doi: 10.1038/s41598-025-21126-y

Figure Lengend Snippet: MBL exposure enhances catabolic processes in EPS-stimulated C2C12 myotubes. ( A ) Representative western blots of autophagy-associated p-ULK1 ser555 , t-ULK1, lysosomal-associated membrane protein 1 (LAMP1), LAMP2, microtubule-associated protein 1 A/1B-light chain 3 (LC3), mitophagy-associated parkin and Bcl-2 interacting protein 3 (BNIP3), and protease calpain 3 protein content. Graphical summaries of ( B ) P-ULK1 ser555 and t-ULK1, ( C ) LAMP1 and LAMP2, ( D ) LC3I, LC3II, and the ratio of LC3II/I, ( E ) parkin, ( F ) and BNIP3 protein content post-EPS. ( G ) Capn3 mRNA expression in C2C12 myotubes post-EPS. ( H ) Calpain 3 protein expression in C2C12 myotubes post-EPS. ( I ) Representative immunofluorescent images of calpain 3 immediately after as well as 12- and 24-hrs post-EPS with and without MBL exposure. Scale bar = 200 μm. ( J ) Mean calpain 3 immunofluorescent intensities within myotube-associated nuclei (myonuclear) as well as within myotubes (myotubular). Calpain 3 (red) to denote myotubes. N = 3. Data are expressed as means ± SEM. * p < 0.05 effect of MBL, # p < 0.05 effect of time, two-way ANOVA. Original blots are presented in Supplementary Fig. 3.

Article Snippet: Clpn3 , Applied Biosystems , 1,925,544 , Mm00482985_m1.

Techniques: Western Blot, Membrane, Expressing

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Production of an antibody recognizing an autoprocessing site within the IS1 region of human CAPN3 . A , schematic illustration showing autoprocessing of CAPN3 and a synthesized peptide used for rabbit immunization. CBSW, calpain-type β-sandwich. IS, internal sequence. NS, N-terminal sequence. PC, protease core. PEF, penta EF motif. C129, H334, and N358 are catalytic centers. One, two, and three indicate the cleavage sites within the IS1 region. The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP). B , immunostaining of HEK293T cells transiently expressing EGFP-CAPN3 or CAPN3:C129S (CS) with anti-AIS1 ( magenta ). EGFP ( green ). DAPI ( blue ). C , western blot of the cell lysates of HEK293T cells transiently expressing EGFP-CAPN3 and CAPN3:CS with anti-CAPN3 ( upper panel) and anti-AIS1 ( lower panel) antibody. An arrow shows the full-length of EGFP-CAPN3CS (130 kDa), and an arrowhead indicates the 58 kDa autocleaved fragment of EGFP-CAPN3. The 130 kDa band of the full-length of EGFP-CAPN3 is faint, because of rapid autolysis. D , schematic illustration of autolytic fragments and the recognition site of anti-AIS1 antibody. The two fragments (a and b) recognized by the anti-AIS1 antibody are approximately 30 kDa. AIS1, autolytic site within IS1; DAPI, 4′,6-diamidino-2-phenylindole; CAPN3, calpain 3.

Article Snippet: Therefore, we examined the quality of the CAPN3 antibody (Proteintech 28476-1-AP) used for immunostaining before examining the subcellular localization of CAPN3 in myotubes.

Techniques: Synthesized, Sequencing, Immunostaining, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Analysis of autolytic activity of LGMDR1 mutants by anti-AIS1 antibody . A , immunostaining of COS-7 cells expressing WT CAPN3 and its mutants with CF488-conjugated anti-AIS1 ( green ) and CF555-conjugated anti-CAPN3 ( red ) antibody. The scale bar represents 20 μm. B , scatter plot of CAPN3 versus AIS1 intensities of WT CAPN3 and its mutants. C , mean ratio of AIS1 to CAPN3 intensity of WT and its mutants. N = 5 ∼ 14 cells. Each plot represents an individual cell value. Mean ± SD. ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Therefore, we examined the quality of the CAPN3 antibody (Proteintech 28476-1-AP) used for immunostaining before examining the subcellular localization of CAPN3 in myotubes.

Techniques: Activity Assay, Immunostaining, Expressing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain Ca 2+ dependently activates CAPN3:S606L in HeLa cells . A , immunostaining of HeLa cells transiently expressing CAPN3:S606L with anti-AIS1 ( green ) and anti-CAPN3 ( magenta ) antibody upon 1 mM ouabain stimulation. The scale bar represents 10 μm. B , change in cytosolic AIS1/CAPN3 immuno-intensity ratio of CAPN3:S606L-expressing cells after ouabain stimulation. Mean ± SD. Number of cells examined are 43, 23, and 52 for 0, 30, and 60 min, respectively. ∗∗∗ p < 0.001, one-way ANOVA with Bonferroni’s test for multiple comparison. C , Ca 2+ imaging of HeLa cells without BSS or with ouabain (1 mM) stimulation with a Ca 2+ indicator, Fura 2. Ratio change of Fura 2 (340 nm/380 nm) is indicated. As a reference, Ca 2+ signals of HeLa cells upon 10 μM ATP stimulation are also indicated. D , ratio change of Fura 2 (340 nm/380 nm) between 0 and 30 min with or without 1 mM ouabain stimulation. Mean ± SD. ∗∗∗ p = 2.45 x 10 -9 , Two tailed student’s unpaired t test. As a reference, peak amplitude of the ratio changes of Fura 2 upon 10 μM ATP stimulation is also included in the figure. N = 123, 103, and 72 cells for BSS, ouabain, and ATP, respectively. E , pretreatment with a Ca 2+ chelator BAPTA-AM (25 μM) inhibits the ouabain-induced autolysis of CAPN3:S606L in HeLa cells. The same membrane was probed with antibodies for CAPN3, AIS1, and β−actin. In the CAPN3 panel, an arrow shows the full-length of CAPN3:S606L (94 kDa), and an arrowhead indicates the autocleaved 58 kDa fragment of CAPN3:S606L. F , increase in the AIS1 band intensity of CAPN3:S606L after ouabain stimulation and its inhibition by BAPTA-AM treatment. The experiments were performed 8, 6, 8, and 4 times for each bar, respectively. Mean ± SD. ∗∗ p = 0.01445, ∗∗∗ p = 0.0037, Steel-Dwass test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Therefore, we examined the quality of the CAPN3 antibody (Proteintech 28476-1-AP) used for immunostaining before examining the subcellular localization of CAPN3 in myotubes.

Techniques: Immunostaining, Expressing, Comparison, Imaging, Two Tailed Test, Membrane, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain increases the autolysis of endogenous CAPN3 in mouse-cultured skeletal muscles . A , autolysis of CAPN3 in cultured skeletal muscles (day 7 in vitro after differentiation) from WT, inactive-form knock-in (KI), and KO mice. The same membrane was probed with antibodies for CAPN3, AIS1, and GAPDH. In the upper CAPN3 panel, an arrow shows the full-length of CAPN3 (94 kDa) and an arrowhead indicates autocleaved 58 kDa fragment of CAPN3. B , changes in the relative band intensity of 94 kDa, 58 kDa, and 30 kDa in A . Mean ± SD. N = 3∼4. ∗ p < 0.05, ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison tes t . C , validation of the specificity of the anti-CAPN3 (Proteintech, 28476-1-AP) antibody on cultured CAPN3 KO skeletal myotubes. Green : CAPN3; magenta : Actinin (Z-band). The scale bar represents 10 μm. D , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the panels of C . Upper panel: WT myotubes; lower panel: KO myotubes. Arrows indicate CAPN3 immunosignals at the M-bands. E , immunohistochemistry of EDLs from WT and CAPN3 KO mice with anti-CPAN3 ( green ) and anti-actinin ( magenta , Z-band) antibody. The scale bar represents 20 μm. F , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the left panel E. Upper panel: WT EDL; lower panel: KO EDL. Arrows indicate CAPN3 immunosignals at the M-bands. AIS1, autolytic site within IS1; CAPN3, calpain 3; EDL, extensor digitorum longus.

Article Snippet: Therefore, we examined the quality of the CAPN3 antibody (Proteintech 28476-1-AP) used for immunostaining before examining the subcellular localization of CAPN3 in myotubes.

Techniques: Cell Culture, Muscles, In Vitro, Knock-In, Membrane, Comparison, Biomarker Discovery, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Translocation of WT but not inactive-form of CAPN3 from the M-bands into the cytosol in cultured skeletal muscles after ouabain stimulation . A and B , immunostaining of AIS1 and CAPN3 in cultured skeletal muscles from WT, KI, and KO mice before ( A ) and aftr 1 mM ouabain stimulation for 60 min ( B ). AIS1 ( green ), CAPN3 ( magenta ), and DAPI ( blue ). The squares show the magnified areas from the images. Arrows show faint striatal patterns of AIS1 signals in ouabain-treated WT myotubes. The scale bar represents 10 μm. C , ouabain-induced cytosolic Ca 2+ levels in WT skeletal myotubes. Fura 2 ratio (340 nm/380 nm) for 60 min was plotted. D , time-dependent increase of Fura 2 ratio (340 nm/380 nm) following 1 mM ouabain stimulation. Mean ± SD. Number of cells analyzed were 28. ∗ p = 0.0229, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Therefore, we examined the quality of the CAPN3 antibody (Proteintech 28476-1-AP) used for immunostaining before examining the subcellular localization of CAPN3 in myotubes.

Techniques: Translocation Assay, Cell Culture, Muscles, Immunostaining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Digestion of spectrin and talin by activated CAPN3 in cultured skeletal muscles upon ouabain stimulation . A , time-dependent increase in the spectrin fragment (∼150 kDa) of cultured skeletal muscles from WT, but not KI and KO mice upon 1 mM ouabain stimulation. Upper arrow indicates the full-length of spectrin, whereas lower arrowhead indicates the cleaved 150 kDa fragment ( upper panel). The same membrane was probed with antibodies for GAPDH ( lower panel ). B , relative band intensities of the 150 kDa spectrin fragments in ( A ). Mean ± SD. Number of experiments were 3 ∼ 4. ∗∗ p < 0.01, one-way ANOVA with Dunnett’s multiple comparison test. C , time-dependent cleavage of talin in WT myotubes by ouabain. The same membrane was probed with antibodies for talin ( upper panel) and GAPDH ( lower panel). The upper arrow shows full length of talin and the lower arrowhead indicates the cleaved talin. D , relative intensity of the cleaved band (∼250 kDa) of talin. Mean ± SD. Number of experiments were 4 ∼ 6. ∗ p < 0.05, Steel-Dwass test. E , expression of Filamin C in the cell lysates of ouabain-treated WT and KI myotubes. Samples were electrophoresed on a 6.0% SDS-PAGE and continued running for an additional 60 min after the dye front reached the bottom of the gels, as previously reported . Top and bottom indicated the top and bottom of the gel, respectively. F , immunoblot of CAPN1 in the cell lysates of ouabain-treated WT and KI myotubes. Autolysis of CAPN1 was not observed in ouabain-treated myotubes. The same membrane was probed with antibody against GAPDH ( lower panel). The experiments were performed three times. CAPN3, calpain 3.

Article Snippet: Therefore, we examined the quality of the CAPN3 antibody (Proteintech 28476-1-AP) used for immunostaining before examining the subcellular localization of CAPN3 in myotubes.

Techniques: Cell Culture, Muscles, Membrane, Comparison, Expressing, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Production of an antibody recognizing an autoprocessing site within the IS1 region of human CAPN3 . A , schematic illustration showing autoprocessing of CAPN3 and a synthesized peptide used for rabbit immunization. CBSW, calpain-type β-sandwich. IS, internal sequence. NS, N-terminal sequence. PC, protease core. PEF, penta EF motif. C129, H334, and N358 are catalytic centers. One, two, and three indicate the cleavage sites within the IS1 region. The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP). B , immunostaining of HEK293T cells transiently expressing EGFP-CAPN3 or CAPN3:C129S (CS) with anti-AIS1 ( magenta ). EGFP ( green ). DAPI ( blue ). C , western blot of the cell lysates of HEK293T cells transiently expressing EGFP-CAPN3 and CAPN3:CS with anti-CAPN3 ( upper panel) and anti-AIS1 ( lower panel) antibody. An arrow shows the full-length of EGFP-CAPN3CS (130 kDa), and an arrowhead indicates the 58 kDa autocleaved fragment of EGFP-CAPN3. The 130 kDa band of the full-length of EGFP-CAPN3 is faint, because of rapid autolysis. D , schematic illustration of autolytic fragments and the recognition site of anti-AIS1 antibody. The two fragments (a and b) recognized by the anti-AIS1 antibody are approximately 30 kDa. AIS1, autolytic site within IS1; DAPI, 4′,6-diamidino-2-phenylindole; CAPN3, calpain 3.

Article Snippet: Rabbit anti-AIS1 and rabbit anti-CAPN3 antibodies (Proteintech, 28476-1-AP) were cross-linked to a fluorescent dye (CF Dye) using Mix-n-Stain CF488A Antibody Labeling Kit (Biotium, Inc, 92253) and Mix-n-Stain CF555 Antibody Labeling Kit (Biotium, Inc, 92254), respectively, according to the manufacturer’s instructions.

Techniques: Synthesized, Sequencing, Immunostaining, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Analysis of autolytic activity of LGMDR1 mutants by anti-AIS1 antibody . A , immunostaining of COS-7 cells expressing WT CAPN3 and its mutants with CF488-conjugated anti-AIS1 ( green ) and CF555-conjugated anti-CAPN3 ( red ) antibody. The scale bar represents 20 μm. B , scatter plot of CAPN3 versus AIS1 intensities of WT CAPN3 and its mutants. C , mean ratio of AIS1 to CAPN3 intensity of WT and its mutants. N = 5 ∼ 14 cells. Each plot represents an individual cell value. Mean ± SD. ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Rabbit anti-AIS1 and rabbit anti-CAPN3 antibodies (Proteintech, 28476-1-AP) were cross-linked to a fluorescent dye (CF Dye) using Mix-n-Stain CF488A Antibody Labeling Kit (Biotium, Inc, 92253) and Mix-n-Stain CF555 Antibody Labeling Kit (Biotium, Inc, 92254), respectively, according to the manufacturer’s instructions.

Techniques: Activity Assay, Immunostaining, Expressing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain Ca 2+ dependently activates CAPN3:S606L in HeLa cells . A , immunostaining of HeLa cells transiently expressing CAPN3:S606L with anti-AIS1 ( green ) and anti-CAPN3 ( magenta ) antibody upon 1 mM ouabain stimulation. The scale bar represents 10 μm. B , change in cytosolic AIS1/CAPN3 immuno-intensity ratio of CAPN3:S606L-expressing cells after ouabain stimulation. Mean ± SD. Number of cells examined are 43, 23, and 52 for 0, 30, and 60 min, respectively. ∗∗∗ p < 0.001, one-way ANOVA with Bonferroni’s test for multiple comparison. C , Ca 2+ imaging of HeLa cells without BSS or with ouabain (1 mM) stimulation with a Ca 2+ indicator, Fura 2. Ratio change of Fura 2 (340 nm/380 nm) is indicated. As a reference, Ca 2+ signals of HeLa cells upon 10 μM ATP stimulation are also indicated. D , ratio change of Fura 2 (340 nm/380 nm) between 0 and 30 min with or without 1 mM ouabain stimulation. Mean ± SD. ∗∗∗ p = 2.45 x 10 -9 , Two tailed student’s unpaired t test. As a reference, peak amplitude of the ratio changes of Fura 2 upon 10 μM ATP stimulation is also included in the figure. N = 123, 103, and 72 cells for BSS, ouabain, and ATP, respectively. E , pretreatment with a Ca 2+ chelator BAPTA-AM (25 μM) inhibits the ouabain-induced autolysis of CAPN3:S606L in HeLa cells. The same membrane was probed with antibodies for CAPN3, AIS1, and β−actin. In the CAPN3 panel, an arrow shows the full-length of CAPN3:S606L (94 kDa), and an arrowhead indicates the autocleaved 58 kDa fragment of CAPN3:S606L. F , increase in the AIS1 band intensity of CAPN3:S606L after ouabain stimulation and its inhibition by BAPTA-AM treatment. The experiments were performed 8, 6, 8, and 4 times for each bar, respectively. Mean ± SD. ∗∗ p = 0.01445, ∗∗∗ p = 0.0037, Steel-Dwass test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Rabbit anti-AIS1 and rabbit anti-CAPN3 antibodies (Proteintech, 28476-1-AP) were cross-linked to a fluorescent dye (CF Dye) using Mix-n-Stain CF488A Antibody Labeling Kit (Biotium, Inc, 92253) and Mix-n-Stain CF555 Antibody Labeling Kit (Biotium, Inc, 92254), respectively, according to the manufacturer’s instructions.

Techniques: Immunostaining, Expressing, Comparison, Imaging, Two Tailed Test, Membrane, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain increases the autolysis of endogenous CAPN3 in mouse-cultured skeletal muscles . A , autolysis of CAPN3 in cultured skeletal muscles (day 7 in vitro after differentiation) from WT, inactive-form knock-in (KI), and KO mice. The same membrane was probed with antibodies for CAPN3, AIS1, and GAPDH. In the upper CAPN3 panel, an arrow shows the full-length of CAPN3 (94 kDa) and an arrowhead indicates autocleaved 58 kDa fragment of CAPN3. B , changes in the relative band intensity of 94 kDa, 58 kDa, and 30 kDa in A . Mean ± SD. N = 3∼4. ∗ p < 0.05, ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison tes t . C , validation of the specificity of the anti-CAPN3 (Proteintech, 28476-1-AP) antibody on cultured CAPN3 KO skeletal myotubes. Green : CAPN3; magenta : Actinin (Z-band). The scale bar represents 10 μm. D , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the panels of C . Upper panel: WT myotubes; lower panel: KO myotubes. Arrows indicate CAPN3 immunosignals at the M-bands. E , immunohistochemistry of EDLs from WT and CAPN3 KO mice with anti-CPAN3 ( green ) and anti-actinin ( magenta , Z-band) antibody. The scale bar represents 20 μm. F , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the left panel E. Upper panel: WT EDL; lower panel: KO EDL. Arrows indicate CAPN3 immunosignals at the M-bands. AIS1, autolytic site within IS1; CAPN3, calpain 3; EDL, extensor digitorum longus.

Article Snippet: Rabbit anti-AIS1 and rabbit anti-CAPN3 antibodies (Proteintech, 28476-1-AP) were cross-linked to a fluorescent dye (CF Dye) using Mix-n-Stain CF488A Antibody Labeling Kit (Biotium, Inc, 92253) and Mix-n-Stain CF555 Antibody Labeling Kit (Biotium, Inc, 92254), respectively, according to the manufacturer’s instructions.

Techniques: Cell Culture, Muscles, In Vitro, Knock-In, Membrane, Comparison, Biomarker Discovery, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Translocation of WT but not inactive-form of CAPN3 from the M-bands into the cytosol in cultured skeletal muscles after ouabain stimulation . A and B , immunostaining of AIS1 and CAPN3 in cultured skeletal muscles from WT, KI, and KO mice before ( A ) and aftr 1 mM ouabain stimulation for 60 min ( B ). AIS1 ( green ), CAPN3 ( magenta ), and DAPI ( blue ). The squares show the magnified areas from the images. Arrows show faint striatal patterns of AIS1 signals in ouabain-treated WT myotubes. The scale bar represents 10 μm. C , ouabain-induced cytosolic Ca 2+ levels in WT skeletal myotubes. Fura 2 ratio (340 nm/380 nm) for 60 min was plotted. D , time-dependent increase of Fura 2 ratio (340 nm/380 nm) following 1 mM ouabain stimulation. Mean ± SD. Number of cells analyzed were 28. ∗ p = 0.0229, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Rabbit anti-AIS1 and rabbit anti-CAPN3 antibodies (Proteintech, 28476-1-AP) were cross-linked to a fluorescent dye (CF Dye) using Mix-n-Stain CF488A Antibody Labeling Kit (Biotium, Inc, 92253) and Mix-n-Stain CF555 Antibody Labeling Kit (Biotium, Inc, 92254), respectively, according to the manufacturer’s instructions.

Techniques: Translocation Assay, Cell Culture, Muscles, Immunostaining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Digestion of spectrin and talin by activated CAPN3 in cultured skeletal muscles upon ouabain stimulation . A , time-dependent increase in the spectrin fragment (∼150 kDa) of cultured skeletal muscles from WT, but not KI and KO mice upon 1 mM ouabain stimulation. Upper arrow indicates the full-length of spectrin, whereas lower arrowhead indicates the cleaved 150 kDa fragment ( upper panel). The same membrane was probed with antibodies for GAPDH ( lower panel ). B , relative band intensities of the 150 kDa spectrin fragments in ( A ). Mean ± SD. Number of experiments were 3 ∼ 4. ∗∗ p < 0.01, one-way ANOVA with Dunnett’s multiple comparison test. C , time-dependent cleavage of talin in WT myotubes by ouabain. The same membrane was probed with antibodies for talin ( upper panel) and GAPDH ( lower panel). The upper arrow shows full length of talin and the lower arrowhead indicates the cleaved talin. D , relative intensity of the cleaved band (∼250 kDa) of talin. Mean ± SD. Number of experiments were 4 ∼ 6. ∗ p < 0.05, Steel-Dwass test. E , expression of Filamin C in the cell lysates of ouabain-treated WT and KI myotubes. Samples were electrophoresed on a 6.0% SDS-PAGE and continued running for an additional 60 min after the dye front reached the bottom of the gels, as previously reported . Top and bottom indicated the top and bottom of the gel, respectively. F , immunoblot of CAPN1 in the cell lysates of ouabain-treated WT and KI myotubes. Autolysis of CAPN1 was not observed in ouabain-treated myotubes. The same membrane was probed with antibody against GAPDH ( lower panel). The experiments were performed three times. CAPN3, calpain 3.

Article Snippet: Rabbit anti-AIS1 and rabbit anti-CAPN3 antibodies (Proteintech, 28476-1-AP) were cross-linked to a fluorescent dye (CF Dye) using Mix-n-Stain CF488A Antibody Labeling Kit (Biotium, Inc, 92253) and Mix-n-Stain CF555 Antibody Labeling Kit (Biotium, Inc, 92254), respectively, according to the manufacturer’s instructions.

Techniques: Cell Culture, Muscles, Membrane, Comparison, Expressing, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Production of an antibody recognizing an autoprocessing site within the IS1 region of human CAPN3 . A , schematic illustration showing autoprocessing of CAPN3 and a synthesized peptide used for rabbit immunization. CBSW, calpain-type β-sandwich. IS, internal sequence. NS, N-terminal sequence. PC, protease core. PEF, penta EF motif. C129, H334, and N358 are catalytic centers. One, two, and three indicate the cleavage sites within the IS1 region. The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP). B , immunostaining of HEK293T cells transiently expressing EGFP-CAPN3 or CAPN3:C129S (CS) with anti-AIS1 ( magenta ). EGFP ( green ). DAPI ( blue ). C , western blot of the cell lysates of HEK293T cells transiently expressing EGFP-CAPN3 and CAPN3:CS with anti-CAPN3 ( upper panel) and anti-AIS1 ( lower panel) antibody. An arrow shows the full-length of EGFP-CAPN3CS (130 kDa), and an arrowhead indicates the 58 kDa autocleaved fragment of EGFP-CAPN3. The 130 kDa band of the full-length of EGFP-CAPN3 is faint, because of rapid autolysis. D , schematic illustration of autolytic fragments and the recognition site of anti-AIS1 antibody. The two fragments (a and b) recognized by the anti-AIS1 antibody are approximately 30 kDa. AIS1, autolytic site within IS1; DAPI, 4′,6-diamidino-2-phenylindole; CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma-Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 °C overnight.

Techniques: Synthesized, Sequencing, Immunostaining, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Analysis of autolytic activity of LGMDR1 mutants by anti-AIS1 antibody . A , immunostaining of COS-7 cells expressing WT CAPN3 and its mutants with CF488-conjugated anti-AIS1 ( green ) and CF555-conjugated anti-CAPN3 ( red ) antibody. The scale bar represents 20 μm. B , scatter plot of CAPN3 versus AIS1 intensities of WT CAPN3 and its mutants. C , mean ratio of AIS1 to CAPN3 intensity of WT and its mutants. N = 5 ∼ 14 cells. Each plot represents an individual cell value. Mean ± SD. ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma-Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 °C overnight.

Techniques: Activity Assay, Immunostaining, Expressing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain Ca 2+ dependently activates CAPN3:S606L in HeLa cells . A , immunostaining of HeLa cells transiently expressing CAPN3:S606L with anti-AIS1 ( green ) and anti-CAPN3 ( magenta ) antibody upon 1 mM ouabain stimulation. The scale bar represents 10 μm. B , change in cytosolic AIS1/CAPN3 immuno-intensity ratio of CAPN3:S606L-expressing cells after ouabain stimulation. Mean ± SD. Number of cells examined are 43, 23, and 52 for 0, 30, and 60 min, respectively. ∗∗∗ p < 0.001, one-way ANOVA with Bonferroni’s test for multiple comparison. C , Ca 2+ imaging of HeLa cells without BSS or with ouabain (1 mM) stimulation with a Ca 2+ indicator, Fura 2. Ratio change of Fura 2 (340 nm/380 nm) is indicated. As a reference, Ca 2+ signals of HeLa cells upon 10 μM ATP stimulation are also indicated. D , ratio change of Fura 2 (340 nm/380 nm) between 0 and 30 min with or without 1 mM ouabain stimulation. Mean ± SD. ∗∗∗ p = 2.45 x 10 -9 , Two tailed student’s unpaired t test. As a reference, peak amplitude of the ratio changes of Fura 2 upon 10 μM ATP stimulation is also included in the figure. N = 123, 103, and 72 cells for BSS, ouabain, and ATP, respectively. E , pretreatment with a Ca 2+ chelator BAPTA-AM (25 μM) inhibits the ouabain-induced autolysis of CAPN3:S606L in HeLa cells. The same membrane was probed with antibodies for CAPN3, AIS1, and β−actin. In the CAPN3 panel, an arrow shows the full-length of CAPN3:S606L (94 kDa), and an arrowhead indicates the autocleaved 58 kDa fragment of CAPN3:S606L. F , increase in the AIS1 band intensity of CAPN3:S606L after ouabain stimulation and its inhibition by BAPTA-AM treatment. The experiments were performed 8, 6, 8, and 4 times for each bar, respectively. Mean ± SD. ∗∗ p = 0.01445, ∗∗∗ p = 0.0037, Steel-Dwass test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma-Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 °C overnight.

Techniques: Immunostaining, Expressing, Comparison, Imaging, Two Tailed Test, Membrane, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain increases the autolysis of endogenous CAPN3 in mouse-cultured skeletal muscles . A , autolysis of CAPN3 in cultured skeletal muscles (day 7 in vitro after differentiation) from WT, inactive-form knock-in (KI), and KO mice. The same membrane was probed with antibodies for CAPN3, AIS1, and GAPDH. In the upper CAPN3 panel, an arrow shows the full-length of CAPN3 (94 kDa) and an arrowhead indicates autocleaved 58 kDa fragment of CAPN3. B , changes in the relative band intensity of 94 kDa, 58 kDa, and 30 kDa in A . Mean ± SD. N = 3∼4. ∗ p < 0.05, ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison tes t . C , validation of the specificity of the anti-CAPN3 (Proteintech, 28476-1-AP) antibody on cultured CAPN3 KO skeletal myotubes. Green : CAPN3; magenta : Actinin (Z-band). The scale bar represents 10 μm. D , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the panels of C . Upper panel: WT myotubes; lower panel: KO myotubes. Arrows indicate CAPN3 immunosignals at the M-bands. E , immunohistochemistry of EDLs from WT and CAPN3 KO mice with anti-CPAN3 ( green ) and anti-actinin ( magenta , Z-band) antibody. The scale bar represents 20 μm. F , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the left panel E. Upper panel: WT EDL; lower panel: KO EDL. Arrows indicate CAPN3 immunosignals at the M-bands. AIS1, autolytic site within IS1; CAPN3, calpain 3; EDL, extensor digitorum longus.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma-Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 °C overnight.

Techniques: Cell Culture, Muscles, In Vitro, Knock-In, Membrane, Comparison, Biomarker Discovery, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Translocation of WT but not inactive-form of CAPN3 from the M-bands into the cytosol in cultured skeletal muscles after ouabain stimulation . A and B , immunostaining of AIS1 and CAPN3 in cultured skeletal muscles from WT, KI, and KO mice before ( A ) and aftr 1 mM ouabain stimulation for 60 min ( B ). AIS1 ( green ), CAPN3 ( magenta ), and DAPI ( blue ). The squares show the magnified areas from the images. Arrows show faint striatal patterns of AIS1 signals in ouabain-treated WT myotubes. The scale bar represents 10 μm. C , ouabain-induced cytosolic Ca 2+ levels in WT skeletal myotubes. Fura 2 ratio (340 nm/380 nm) for 60 min was plotted. D , time-dependent increase of Fura 2 ratio (340 nm/380 nm) following 1 mM ouabain stimulation. Mean ± SD. Number of cells analyzed were 28. ∗ p = 0.0229, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma-Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 °C overnight.

Techniques: Translocation Assay, Cell Culture, Muscles, Immunostaining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Digestion of spectrin and talin by activated CAPN3 in cultured skeletal muscles upon ouabain stimulation . A , time-dependent increase in the spectrin fragment (∼150 kDa) of cultured skeletal muscles from WT, but not KI and KO mice upon 1 mM ouabain stimulation. Upper arrow indicates the full-length of spectrin, whereas lower arrowhead indicates the cleaved 150 kDa fragment ( upper panel). The same membrane was probed with antibodies for GAPDH ( lower panel ). B , relative band intensities of the 150 kDa spectrin fragments in ( A ). Mean ± SD. Number of experiments were 3 ∼ 4. ∗∗ p < 0.01, one-way ANOVA with Dunnett’s multiple comparison test. C , time-dependent cleavage of talin in WT myotubes by ouabain. The same membrane was probed with antibodies for talin ( upper panel) and GAPDH ( lower panel). The upper arrow shows full length of talin and the lower arrowhead indicates the cleaved talin. D , relative intensity of the cleaved band (∼250 kDa) of talin. Mean ± SD. Number of experiments were 4 ∼ 6. ∗ p < 0.05, Steel-Dwass test. E , expression of Filamin C in the cell lysates of ouabain-treated WT and KI myotubes. Samples were electrophoresed on a 6.0% SDS-PAGE and continued running for an additional 60 min after the dye front reached the bottom of the gels, as previously reported . Top and bottom indicated the top and bottom of the gel, respectively. F , immunoblot of CAPN1 in the cell lysates of ouabain-treated WT and KI myotubes. Autolysis of CAPN1 was not observed in ouabain-treated myotubes. The same membrane was probed with antibody against GAPDH ( lower panel). The experiments were performed three times. CAPN3, calpain 3.

Article Snippet: Then, the sections were probed with appropriate antibodies (anti-CAPN3 antibody (Proteintech, 28476-1-AP, 1:500); anti-actinin (Sigma-Aldrich, EA-53, 1:500)) diluted in the appropriate blocking buffer at 4 °C overnight.

Techniques: Cell Culture, Muscles, Membrane, Comparison, Expressing, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Production of an antibody recognizing an autoprocessing site within the IS1 region of human CAPN3 . A , schematic illustration showing autoprocessing of CAPN3 and a synthesized peptide used for rabbit immunization. CBSW, calpain-type β-sandwich. IS, internal sequence. NS, N-terminal sequence. PC, protease core. PEF, penta EF motif. C129, H334, and N358 are catalytic centers. One, two, and three indicate the cleavage sites within the IS1 region. The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP). B , immunostaining of HEK293T cells transiently expressing EGFP-CAPN3 or CAPN3:C129S (CS) with anti-AIS1 ( magenta ). EGFP ( green ). DAPI ( blue ). C , western blot of the cell lysates of HEK293T cells transiently expressing EGFP-CAPN3 and CAPN3:CS with anti-CAPN3 ( upper panel) and anti-AIS1 ( lower panel) antibody. An arrow shows the full-length of EGFP-CAPN3CS (130 kDa), and an arrowhead indicates the 58 kDa autocleaved fragment of EGFP-CAPN3. The 130 kDa band of the full-length of EGFP-CAPN3 is faint, because of rapid autolysis. D , schematic illustration of autolytic fragments and the recognition site of anti-AIS1 antibody. The two fragments (a and b) recognized by the anti-AIS1 antibody are approximately 30 kDa. AIS1, autolytic site within IS1; DAPI, 4′,6-diamidino-2-phenylindole; CAPN3, calpain 3.

Article Snippet: The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP).

Techniques: Synthesized, Sequencing, Immunostaining, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Analysis of autolytic activity of LGMDR1 mutants by anti-AIS1 antibody . A , immunostaining of COS-7 cells expressing WT CAPN3 and its mutants with CF488-conjugated anti-AIS1 ( green ) and CF555-conjugated anti-CAPN3 ( red ) antibody. The scale bar represents 20 μm. B , scatter plot of CAPN3 versus AIS1 intensities of WT CAPN3 and its mutants. C , mean ratio of AIS1 to CAPN3 intensity of WT and its mutants. N = 5 ∼ 14 cells. Each plot represents an individual cell value. Mean ± SD. ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP).

Techniques: Activity Assay, Immunostaining, Expressing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain Ca 2+ dependently activates CAPN3:S606L in HeLa cells . A , immunostaining of HeLa cells transiently expressing CAPN3:S606L with anti-AIS1 ( green ) and anti-CAPN3 ( magenta ) antibody upon 1 mM ouabain stimulation. The scale bar represents 10 μm. B , change in cytosolic AIS1/CAPN3 immuno-intensity ratio of CAPN3:S606L-expressing cells after ouabain stimulation. Mean ± SD. Number of cells examined are 43, 23, and 52 for 0, 30, and 60 min, respectively. ∗∗∗ p < 0.001, one-way ANOVA with Bonferroni’s test for multiple comparison. C , Ca 2+ imaging of HeLa cells without BSS or with ouabain (1 mM) stimulation with a Ca 2+ indicator, Fura 2. Ratio change of Fura 2 (340 nm/380 nm) is indicated. As a reference, Ca 2+ signals of HeLa cells upon 10 μM ATP stimulation are also indicated. D , ratio change of Fura 2 (340 nm/380 nm) between 0 and 30 min with or without 1 mM ouabain stimulation. Mean ± SD. ∗∗∗ p = 2.45 x 10 -9 , Two tailed student’s unpaired t test. As a reference, peak amplitude of the ratio changes of Fura 2 upon 10 μM ATP stimulation is also included in the figure. N = 123, 103, and 72 cells for BSS, ouabain, and ATP, respectively. E , pretreatment with a Ca 2+ chelator BAPTA-AM (25 μM) inhibits the ouabain-induced autolysis of CAPN3:S606L in HeLa cells. The same membrane was probed with antibodies for CAPN3, AIS1, and β−actin. In the CAPN3 panel, an arrow shows the full-length of CAPN3:S606L (94 kDa), and an arrowhead indicates the autocleaved 58 kDa fragment of CAPN3:S606L. F , increase in the AIS1 band intensity of CAPN3:S606L after ouabain stimulation and its inhibition by BAPTA-AM treatment. The experiments were performed 8, 6, 8, and 4 times for each bar, respectively. Mean ± SD. ∗∗ p = 0.01445, ∗∗∗ p = 0.0037, Steel-Dwass test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP).

Techniques: Immunostaining, Expressing, Comparison, Imaging, Two Tailed Test, Membrane, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain increases the autolysis of endogenous CAPN3 in mouse-cultured skeletal muscles . A , autolysis of CAPN3 in cultured skeletal muscles (day 7 in vitro after differentiation) from WT, inactive-form knock-in (KI), and KO mice. The same membrane was probed with antibodies for CAPN3, AIS1, and GAPDH. In the upper CAPN3 panel, an arrow shows the full-length of CAPN3 (94 kDa) and an arrowhead indicates autocleaved 58 kDa fragment of CAPN3. B , changes in the relative band intensity of 94 kDa, 58 kDa, and 30 kDa in A . Mean ± SD. N = 3∼4. ∗ p < 0.05, ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison tes t . C , validation of the specificity of the anti-CAPN3 (Proteintech, 28476-1-AP) antibody on cultured CAPN3 KO skeletal myotubes. Green : CAPN3; magenta : Actinin (Z-band). The scale bar represents 10 μm. D , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the panels of C . Upper panel: WT myotubes; lower panel: KO myotubes. Arrows indicate CAPN3 immunosignals at the M-bands. E , immunohistochemistry of EDLs from WT and CAPN3 KO mice with anti-CPAN3 ( green ) and anti-actinin ( magenta , Z-band) antibody. The scale bar represents 20 μm. F , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the left panel E. Upper panel: WT EDL; lower panel: KO EDL. Arrows indicate CAPN3 immunosignals at the M-bands. AIS1, autolytic site within IS1; CAPN3, calpain 3; EDL, extensor digitorum longus.

Article Snippet: The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP).

Techniques: Cell Culture, Muscles, In Vitro, Knock-In, Membrane, Comparison, Biomarker Discovery, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Translocation of WT but not inactive-form of CAPN3 from the M-bands into the cytosol in cultured skeletal muscles after ouabain stimulation . A and B , immunostaining of AIS1 and CAPN3 in cultured skeletal muscles from WT, KI, and KO mice before ( A ) and aftr 1 mM ouabain stimulation for 60 min ( B ). AIS1 ( green ), CAPN3 ( magenta ), and DAPI ( blue ). The squares show the magnified areas from the images. Arrows show faint striatal patterns of AIS1 signals in ouabain-treated WT myotubes. The scale bar represents 10 μm. C , ouabain-induced cytosolic Ca 2+ levels in WT skeletal myotubes. Fura 2 ratio (340 nm/380 nm) for 60 min was plotted. D , time-dependent increase of Fura 2 ratio (340 nm/380 nm) following 1 mM ouabain stimulation. Mean ± SD. Number of cells analyzed were 28. ∗ p = 0.0229, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP).

Techniques: Translocation Assay, Cell Culture, Muscles, Immunostaining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Digestion of spectrin and talin by activated CAPN3 in cultured skeletal muscles upon ouabain stimulation . A , time-dependent increase in the spectrin fragment (∼150 kDa) of cultured skeletal muscles from WT, but not KI and KO mice upon 1 mM ouabain stimulation. Upper arrow indicates the full-length of spectrin, whereas lower arrowhead indicates the cleaved 150 kDa fragment ( upper panel). The same membrane was probed with antibodies for GAPDH ( lower panel ). B , relative band intensities of the 150 kDa spectrin fragments in ( A ). Mean ± SD. Number of experiments were 3 ∼ 4. ∗∗ p < 0.01, one-way ANOVA with Dunnett’s multiple comparison test. C , time-dependent cleavage of talin in WT myotubes by ouabain. The same membrane was probed with antibodies for talin ( upper panel) and GAPDH ( lower panel). The upper arrow shows full length of talin and the lower arrowhead indicates the cleaved talin. D , relative intensity of the cleaved band (∼250 kDa) of talin. Mean ± SD. Number of experiments were 4 ∼ 6. ∗ p < 0.05, Steel-Dwass test. E , expression of Filamin C in the cell lysates of ouabain-treated WT and KI myotubes. Samples were electrophoresed on a 6.0% SDS-PAGE and continued running for an additional 60 min after the dye front reached the bottom of the gels, as previously reported . Top and bottom indicated the top and bottom of the gel, respectively. F , immunoblot of CAPN1 in the cell lysates of ouabain-treated WT and KI myotubes. Autolysis of CAPN1 was not observed in ouabain-treated myotubes. The same membrane was probed with antibody against GAPDH ( lower panel). The experiments were performed three times. CAPN3, calpain 3.

Article Snippet: The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP).

Techniques: Cell Culture, Muscles, Membrane, Comparison, Expressing, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Production of an antibody recognizing an autoprocessing site within the IS1 region of human CAPN3 . A , schematic illustration showing autoprocessing of CAPN3 and a synthesized peptide used for rabbit immunization. CBSW, calpain-type β-sandwich. IS, internal sequence. NS, N-terminal sequence. PC, protease core. PEF, penta EF motif. C129, H334, and N358 are catalytic centers. One, two, and three indicate the cleavage sites within the IS1 region. The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 284SS76-1-AP). B , immunostaining of HEK293T cells transiently expressing EGFP-CAPN3 or CAPN3:C129S (CS) with anti-AIS1 ( magenta ). EGFP ( green ). DAPI ( blue ). C , western blot of the cell lysates of HEK293T cells transiently expressing EGFP-CAPN3 and CAPN3:CS with anti-CAPN3 ( upper panel) and anti-AIS1 ( lower panel) antibody. An arrow shows the full-length of EGFP-CAPN3CS (130 kDa), and an arrowhead indicates the 58 kDa autocleaved fragment of EGFP-CAPN3. The 130 kDa band of the full-length of EGFP-CAPN3 is faint, because of rapid autolysis. D , schematic illustration of autolytic fragments and the recognition site of anti-AIS1 antibody. The two fragments (a and b) recognized by the anti-AIS1 antibody are approximately 30 kDa. AIS1, autolytic site within IS1; DAPI, 4′,6-diamidino-2-phenylindole; CAPN3, calpain 3.

Article Snippet: The antibodies used in this study include rabbit anti-CAPN3 polyclonal antibody (Proteintech, 28476-1-AP, antigen: 488–666 aa encoded by BC146672 , 1:1000), anti-α-actinin antibody (Sigma-Aldrich, EA-53, 1: 1000), anti-spectrin alpha chain (nonerythroid) antibody (Sigma-Aldrich, MAB1622, clone AA6, 1:1000), anti-talin antibody (Sigma-Aldrich, T3287, 1:1000), anti-β-actin (MBL, M177–3, 1:1000), and filamin C (Myomedix Ltd, FLC-1, rabbit, 1:1000).

Techniques: Synthesized, Sequencing, Immunostaining, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Analysis of autolytic activity of LGMDR1 mutants by anti-AIS1 antibody . A , immunostaining of COS-7 cells expressing WT CAPN3 and its mutants with CF488-conjugated anti-AIS1 ( green ) and CF555-conjugated anti-CAPN3 ( red ) antibody. The scale bar represents 20 μm. B , scatter plot of CAPN3 versus AIS1 intensities of WT CAPN3 and its mutants. C , mean ratio of AIS1 to CAPN3 intensity of WT and its mutants. N = 5 ∼ 14 cells. Each plot represents an individual cell value. Mean ± SD. ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: The antibodies used in this study include rabbit anti-CAPN3 polyclonal antibody (Proteintech, 28476-1-AP, antigen: 488–666 aa encoded by BC146672 , 1:1000), anti-α-actinin antibody (Sigma-Aldrich, EA-53, 1: 1000), anti-spectrin alpha chain (nonerythroid) antibody (Sigma-Aldrich, MAB1622, clone AA6, 1:1000), anti-talin antibody (Sigma-Aldrich, T3287, 1:1000), anti-β-actin (MBL, M177–3, 1:1000), and filamin C (Myomedix Ltd, FLC-1, rabbit, 1:1000).

Techniques: Activity Assay, Immunostaining, Expressing, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain Ca 2+ dependently activates CAPN3:S606L in HeLa cells . A , immunostaining of HeLa cells transiently expressing CAPN3:S606L with anti-AIS1 ( green ) and anti-CAPN3 ( magenta ) antibody upon 1 mM ouabain stimulation. The scale bar represents 10 μm. B , change in cytosolic AIS1/CAPN3 immuno-intensity ratio of CAPN3:S606L-expressing cells after ouabain stimulation. Mean ± SD. Number of cells examined are 43, 23, and 52 for 0, 30, and 60 min, respectively. ∗∗∗ p < 0.001, one-way ANOVA with Bonferroni’s test for multiple comparison. C , Ca 2+ imaging of HeLa cells without BSS or with ouabain (1 mM) stimulation with a Ca 2+ indicator, Fura 2. Ratio change of Fura 2 (340 nm/380 nm) is indicated. As a reference, Ca 2+ signals of HeLa cells upon 10 μM ATP stimulation are also indicated. D , ratio change of Fura 2 (340 nm/380 nm) between 0 and 30 min with or without 1 mM ouabain stimulation. Mean ± SD. ∗∗∗ p = 2.45 x 10 -9 , Two tailed student’s unpaired t test. As a reference, peak amplitude of the ratio changes of Fura 2 upon 10 μM ATP stimulation is also included in the figure. N = 123, 103, and 72 cells for BSS, ouabain, and ATP, respectively. E , pretreatment with a Ca 2+ chelator BAPTA-AM (25 μM) inhibits the ouabain-induced autolysis of CAPN3:S606L in HeLa cells. The same membrane was probed with antibodies for CAPN3, AIS1, and β−actin. In the CAPN3 panel, an arrow shows the full-length of CAPN3:S606L (94 kDa), and an arrowhead indicates the autocleaved 58 kDa fragment of CAPN3:S606L. F , increase in the AIS1 band intensity of CAPN3:S606L after ouabain stimulation and its inhibition by BAPTA-AM treatment. The experiments were performed 8, 6, 8, and 4 times for each bar, respectively. Mean ± SD. ∗∗ p = 0.01445, ∗∗∗ p = 0.0037, Steel-Dwass test. AIS1, autolytic site within IS1; CAPN3, calpain 3.

Article Snippet: The antibodies used in this study include rabbit anti-CAPN3 polyclonal antibody (Proteintech, 28476-1-AP, antigen: 488–666 aa encoded by BC146672 , 1:1000), anti-α-actinin antibody (Sigma-Aldrich, EA-53, 1: 1000), anti-spectrin alpha chain (nonerythroid) antibody (Sigma-Aldrich, MAB1622, clone AA6, 1:1000), anti-talin antibody (Sigma-Aldrich, T3287, 1:1000), anti-β-actin (MBL, M177–3, 1:1000), and filamin C (Myomedix Ltd, FLC-1, rabbit, 1:1000).

Techniques: Immunostaining, Expressing, Comparison, Imaging, Two Tailed Test, Membrane, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Ouabain increases the autolysis of endogenous CAPN3 in mouse-cultured skeletal muscles . A , autolysis of CAPN3 in cultured skeletal muscles (day 7 in vitro after differentiation) from WT, inactive-form knock-in (KI), and KO mice. The same membrane was probed with antibodies for CAPN3, AIS1, and GAPDH. In the upper CAPN3 panel, an arrow shows the full-length of CAPN3 (94 kDa) and an arrowhead indicates autocleaved 58 kDa fragment of CAPN3. B , changes in the relative band intensity of 94 kDa, 58 kDa, and 30 kDa in A . Mean ± SD. N = 3∼4. ∗ p < 0.05, ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison tes t . C , validation of the specificity of the anti-CAPN3 (Proteintech, 28476-1-AP) antibody on cultured CAPN3 KO skeletal myotubes. Green : CAPN3; magenta : Actinin (Z-band). The scale bar represents 10 μm. D , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the panels of C . Upper panel: WT myotubes; lower panel: KO myotubes. Arrows indicate CAPN3 immunosignals at the M-bands. E , immunohistochemistry of EDLs from WT and CAPN3 KO mice with anti-CPAN3 ( green ) and anti-actinin ( magenta , Z-band) antibody. The scale bar represents 20 μm. F , intensity profiles of CAPN3 and actinin signals on white bars (2 μm thick) in the left panel E. Upper panel: WT EDL; lower panel: KO EDL. Arrows indicate CAPN3 immunosignals at the M-bands. AIS1, autolytic site within IS1; CAPN3, calpain 3; EDL, extensor digitorum longus.

Article Snippet: The antibodies used in this study include rabbit anti-CAPN3 polyclonal antibody (Proteintech, 28476-1-AP, antigen: 488–666 aa encoded by BC146672 , 1:1000), anti-α-actinin antibody (Sigma-Aldrich, EA-53, 1: 1000), anti-spectrin alpha chain (nonerythroid) antibody (Sigma-Aldrich, MAB1622, clone AA6, 1:1000), anti-talin antibody (Sigma-Aldrich, T3287, 1:1000), anti-β-actin (MBL, M177–3, 1:1000), and filamin C (Myomedix Ltd, FLC-1, rabbit, 1:1000).

Techniques: Cell Culture, Muscles, In Vitro, Knock-In, Membrane, Comparison, Biomarker Discovery, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Translocation of WT but not inactive-form of CAPN3 from the M-bands into the cytosol in cultured skeletal muscles after ouabain stimulation . A and B , immunostaining of AIS1 and CAPN3 in cultured skeletal muscles from WT, KI, and KO mice before ( A ) and aftr 1 mM ouabain stimulation for 60 min ( B ). AIS1 ( green ), CAPN3 ( magenta ), and DAPI ( blue ). The squares show the magnified areas from the images. Arrows show faint striatal patterns of AIS1 signals in ouabain-treated WT myotubes. The scale bar represents 10 μm. C , ouabain-induced cytosolic Ca 2+ levels in WT skeletal myotubes. Fura 2 ratio (340 nm/380 nm) for 60 min was plotted. D , time-dependent increase of Fura 2 ratio (340 nm/380 nm) following 1 mM ouabain stimulation. Mean ± SD. Number of cells analyzed were 28. ∗ p = 0.0229, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolytic site within IS1; CAPN3, calpain 3; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The antibodies used in this study include rabbit anti-CAPN3 polyclonal antibody (Proteintech, 28476-1-AP, antigen: 488–666 aa encoded by BC146672 , 1:1000), anti-α-actinin antibody (Sigma-Aldrich, EA-53, 1: 1000), anti-spectrin alpha chain (nonerythroid) antibody (Sigma-Aldrich, MAB1622, clone AA6, 1:1000), anti-talin antibody (Sigma-Aldrich, T3287, 1:1000), anti-β-actin (MBL, M177–3, 1:1000), and filamin C (Myomedix Ltd, FLC-1, rabbit, 1:1000).

Techniques: Translocation Assay, Cell Culture, Muscles, Immunostaining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: In situ detection of activation of CAPN3, a responsible gene product for LGMDR1, in mouse skeletal myotubes

doi: 10.1016/j.jbc.2025.108536

Figure Lengend Snippet: Digestion of spectrin and talin by activated CAPN3 in cultured skeletal muscles upon ouabain stimulation . A , time-dependent increase in the spectrin fragment (∼150 kDa) of cultured skeletal muscles from WT, but not KI and KO mice upon 1 mM ouabain stimulation. Upper arrow indicates the full-length of spectrin, whereas lower arrowhead indicates the cleaved 150 kDa fragment ( upper panel). The same membrane was probed with antibodies for GAPDH ( lower panel ). B , relative band intensities of the 150 kDa spectrin fragments in ( A ). Mean ± SD. Number of experiments were 3 ∼ 4. ∗∗ p < 0.01, one-way ANOVA with Dunnett’s multiple comparison test. C , time-dependent cleavage of talin in WT myotubes by ouabain. The same membrane was probed with antibodies for talin ( upper panel) and GAPDH ( lower panel). The upper arrow shows full length of talin and the lower arrowhead indicates the cleaved talin. D , relative intensity of the cleaved band (∼250 kDa) of talin. Mean ± SD. Number of experiments were 4 ∼ 6. ∗ p < 0.05, Steel-Dwass test. E , expression of Filamin C in the cell lysates of ouabain-treated WT and KI myotubes. Samples were electrophoresed on a 6.0% SDS-PAGE and continued running for an additional 60 min after the dye front reached the bottom of the gels, as previously reported . Top and bottom indicated the top and bottom of the gel, respectively. F , immunoblot of CAPN1 in the cell lysates of ouabain-treated WT and KI myotubes. Autolysis of CAPN1 was not observed in ouabain-treated myotubes. The same membrane was probed with antibody against GAPDH ( lower panel). The experiments were performed three times. CAPN3, calpain 3.

Article Snippet: The antibodies used in this study include rabbit anti-CAPN3 polyclonal antibody (Proteintech, 28476-1-AP, antigen: 488–666 aa encoded by BC146672 , 1:1000), anti-α-actinin antibody (Sigma-Aldrich, EA-53, 1: 1000), anti-spectrin alpha chain (nonerythroid) antibody (Sigma-Aldrich, MAB1622, clone AA6, 1:1000), anti-talin antibody (Sigma-Aldrich, T3287, 1:1000), anti-β-actin (MBL, M177–3, 1:1000), and filamin C (Myomedix Ltd, FLC-1, rabbit, 1:1000).

Techniques: Cell Culture, Muscles, Membrane, Comparison, Expressing, SDS Page, Western Blot