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: Figure 1. 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 b-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, 40,6-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: Figure 2. 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 mm. 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: Figure 3. Ouabain Ca2+ 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 mm. 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, Ca2+ imaging of HeLa cells without BSS or with ouabain (1 mM) stimulation with a Ca2+ indicator, Fura 2. Ratio change of Fura 2 (340 nm/380 nm) is indicated. As a reference, Ca2+ signals of HeLa cells upon 10 mM 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 mM ATP stimulation is also included in the figure. N = 123, 103, and 72 cells for BSS, ouabain, and ATP, respectively. E, pretreatment with a Ca2+ chelator BAPTA- AM (25 mM) inhibits the ouabain-induced autolysis of CAPN3:S606L in HeLa cells. The same membrane was probed with antibodies for CAPN3, AIS1, and b−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: Figure 4. 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 = 34. *p < 0.05, ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. 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 mm. D, intensity profiles of CAPN3 and actinin signals on white bars (2 mm thick) in the panels of C. Upper panel: WT myotubes; lower panel: KO myotubes. Arrows indicate CAPN3 immunosignals at the M-bands. E, immunohis- tochemistry of EDLs from WT and CAPN3 KO mice with anti-CPAN3 (green) and anti-actinin (magenta, Z-band) antibody. The scale bar represents 20 mm. F, intensity profiles of CAPN3 and actinin signals on white bars (2 mm 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

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: Figure 5. 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 mm. C, ouabain-induced cytosolic Ca2+ 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, 40,6-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: Figure 7. 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 (59). 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:

Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

doi: 10.1128/MCB.22.8.2716-2727.2002

Figure Lengend Snippet: EGF-induced calpain activation. NR6WT cells were plated on tissue culture chamber slides (Nunc) and made quiescent for 24 h in MEMα with 0.5% dialyzed fetal calf serum. Cells were treated or not with CPT-cAMPS (1 μM), Rp-8Br-cAMPS (5 μM), and/or Rp-8Br-cGMPS (5 μM) 30 min prior to EGF (10 nM) treatment in the presence of BOC-LM-CMAC (Molecular Probes). Then cells were treated or not with EGF (10 nM) for 5 min. Calpain activation was assessed by fluorescence microscopy. The fluorescence indicates calpain activity. The panel for nontreated control cells is labeled as nTx. The pictures shown are representative of n = 9.

Article Snippet: Cells were treated or not with dexamethasone (2 μM) for 18 h. Cells were lysed, and equal amount of proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and immunoblotted with anti-m-calpain antibody (Santa Cruz), anti-GFP antibody (Clontech) (A), or anti-His antibody (Santa Cruz) (B).

Techniques: Activation Assay, Fluorescence, Microscopy, Activity Assay, Labeling

Journal:

Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

doi: 10.1128/MCB.22.8.2716-2727.2002

Figure Lengend Snippet: m-Calpain phosphorylation at ST369/370 by PKA limits proteolytic activity. Both His-tagged wild-type- and ST369AA hCANP-expressing cells were grown and made quiescent for 24 h. Cells were treated with dexamethasone (2 μM) for 18 h to induce expression of exogenous hCANP constructs. To determine whether m-calpain was a target of PKA, hCANP was purified by Ni-NTA affinity chromatography and dialyzed with 50 mM Tris-HCl (pH 7.4) for 24 h. (A) Purified hCANP was treated with constitutively active cAMP-dependent PKA catalytic subunit and [γ-32P]ATP for 15 min. The samples were analyzed by separation through an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was used for autoradiography and subsequently blotted with anti-m-calpain antibody (Santa Cruz). The results shown are representative of two independent experiments. (B) Purified hCANP was incubated with recombinant Tau protein with or without calcium (40 μM) in the presence or absence of ERK (300 U) for 10 min. Samples were analyzed by SDS-10% polyacrylamide gel electrophoresis and transferred to a nylon membrane (Immobilon-P). The transferred membrane was blotted with anti-Tau antibody (Zymed). Reduction in the size of Tau indicates calpain activity. The results shown are representative of two independent experiments. (C) Purified hCANP was incubated with MAP2 in the presence or absence of ERK (300 U) and PKA catalytic subunit (25 U) for 20 min. Samples were analyzed on an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was blotted with anti-MAP2 antibody (Sigma). Loss of cleavage products of 75 to 120 kDa indicated activity. The results shown are representative of two independent experiments.

Article Snippet: Cells were treated or not with dexamethasone (2 μM) for 18 h. Cells were lysed, and equal amount of proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and immunoblotted with anti-m-calpain antibody (Santa Cruz), anti-GFP antibody (Clontech) (A), or anti-His antibody (Santa Cruz) (B).

Techniques: Activity Assay, Expressing, Construct, Purification, Affinity Chromatography, Autoradiography, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis

Journal:

Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

doi: 10.1128/MCB.22.8.2716-2727.2002

Figure Lengend Snippet: hCANP expression in NR6WT cells. MMTV-driven hCANP constructs were introduced into NR6WT cells by electroporation. Polyclonal stable expression cell lines were selected in the presence of hygromycin. Cells were treated or not with dexamethasone (2 μM) for 18 h. Cells were lysed, and equal amount of proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and immunoblotted with anti-m-calpain antibody (Santa Cruz), anti-GFP antibody (Clontech) (A), or anti-His antibody (Santa Cruz) (B). The anticalpain antibody recognizes the endogenous mouse m-calpain (∼80 kDa) as well as the exogenously encoded expressed human calpain-GFP (∼110-kDa) and His (∼80-kDa) fusion proteins. Shown are representative blots from three independent experiments.

Article Snippet: Cells were treated or not with dexamethasone (2 μM) for 18 h. Cells were lysed, and equal amount of proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and immunoblotted with anti-m-calpain antibody (Santa Cruz), anti-GFP antibody (Clontech) (A), or anti-His antibody (Santa Cruz) (B).

Techniques: Expressing, Construct, Electroporation, Polyacrylamide Gel Electrophoresis

Journal:

Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

doi: 10.1128/MCB.22.8.2716-2727.2002

Figure Lengend Snippet: EGF-induced calpain activation in hCANP construct expressing NR6WT cells. NR6WT cells with His-tagged hCANP constructs were plated on tissue culture chamber slides (Nunc) and made quiescent for 24 h in MEMα with 0.5% dialyzed fetal calf serum. Cells were treated with dexamethasone (2 μM) for 18 h. Then cells were treated or not with CPT-cAMPS (1 μM) 30 min prior to EGF (10 nM) treatment in the presence of BOC-LM-CMAC (Molecular Probes). Finally cells were treated or not with EGF (10 nM) for 5 min. Calpain activation was assessed by fluorescence microscopy. The fluorescence indicates calpain activity. nTx, nontreated control cells. The pictures shown are representative of n = 9.

Article Snippet: Cells were treated or not with dexamethasone (2 μM) for 18 h. Cells were lysed, and equal amount of proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and immunoblotted with anti-m-calpain antibody (Santa Cruz), anti-GFP antibody (Clontech) (A), or anti-His antibody (Santa Cruz) (B).

Techniques: Activation Assay, Construct, Expressing, Fluorescence, Microscopy, Activity Assay

Journal:

Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

doi: 10.1128/MCB.22.8.2716-2727.2002

Figure Lengend Snippet: EGF-induced cell migration activity of hCANP construct-expressing NR6WT cells. NR6WT cells with His-tagged hCANP constructs were utilized for an in vitro cell migration assay to assess the biological effect of dominant negative m-calpain. Cells were grown to confluence in MEMα with 7.5% fetal calf serum containing 100 μg of hygromycin/ml. Cells were made quiescent with MEMα with 0.5% dialyzed fetal calf serum for 24 h before treatment with EGF. Cells were treated or not with antisense mouse m-calpain to downregulate endogenous mouse m-calpain. Cells were treated with EGF (10 nM) and antisense m-calpain (20 μM) for 6 h. Cells were then incubated for a further 12 to 14 h in quiescent medium with antisense m-calpain. Cells were treated (A and C) or not (B and D) with dexamethasone (2 μM) 2 h prior to EGF treatment. Then cells were treated with CPT-cAMPS (1 μg/ml) and EGF (10 nM). Cell migration activity was measured as the ability of the cells to move into an acellular area after 24 h of EGF treatment. The data for nontreated control cells are labeled as nTx. The data are shown as ratios to 10 nM EGF-induced cell migration in the absence of antisense m-calpain oligonucleotide. The data are the means ± SEM of more than three independent studies, each performed in triplicate. Statistical analysis was performed by Student's t test. Cell lysates analyzed by immunoblotting (Santa Cruz) demonstrated downregulation of endogenous calpain (E). The results shown are representative of two independent experiments. n.s., not significant.

Article Snippet: Cells were treated or not with dexamethasone (2 μM) for 18 h. Cells were lysed, and equal amount of proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and immunoblotted with anti-m-calpain antibody (Santa Cruz), anti-GFP antibody (Clontech) (A), or anti-His antibody (Santa Cruz) (B).

Techniques: Migration, Activity Assay, Construct, Expressing, In Vitro, Cell Migration Assay, Dominant Negative Mutation, Incubation, Labeling, Western Blot

Journal:

Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

doi: 10.1128/MCB.22.8.2716-2727.2002

Figure Lengend Snippet: Ribbon diagram of calpain (A) and close-up view of the S369 region (B). Calpain domains are indicated. The box represents the approximate area which is shown in close-up (B). Interaction distances are also given, along with three key amino acids involved in the interaction upon phosphorylation of S369.

Article Snippet: Cells were treated or not with dexamethasone (2 μM) for 18 h. Cells were lysed, and equal amount of proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and immunoblotted with anti-m-calpain antibody (Santa Cruz), anti-GFP antibody (Clontech) (A), or anti-His antibody (Santa Cruz) (B).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Aberrant mechanical loading induces annulus fibrosus cells apoptosis in intervertebral disc degeneration via mechanosensitive ion channel Piezo1

doi: 10.1186/s13075-023-03093-9

Figure Lengend Snippet: Piezo1 promotes AFCs apoptosis via Ca2 + /Calpain2/Caspase3 pathway. a The activity of Calpains in the Ctrl group, CMS group, CMS + Lv-Ctrl group, and CMS + Lv-Piezo1 group as determined by Calpain activity detection kit ( n = 3). b Western blotting analysis showing the Calpain1, Calpain2, Bax, Cleaved-Caspase3 expression in the Ctrl group, CMS group, CMS + Lv-Ctrl group, and CMS + Lv-Piezo1 group ( n = 3). c Representative flow cytometry scatter plots detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group. d Representative Tunel staining fluorescence pictures detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group (scale bar = 50 μm). Apoptotic AFCs appeared red and nuclei were counterstained with Hoechst 33,358 (blue). e Representative MMP staining fluorescence pictures detecting AFCs apoptosis in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group (scale bar = 50 μm). MMP appeared red and nuclei were counterstained with Hoechst 33,358 (blue). f Statistic data of apoptotic AFCs in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by flow cytometry ( n = 3). g Statistic data of Tunel-positive apoptotic AFCs in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by Tunel staining ( n = 3). h Statistic data of mean MMP fluorescent intensity in AFC in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group determined by MMP staining ( n = 3). i Western blotting analysis showing the Bax, Cleaved-Caspase3 expression in the CMS + Yoda1 group, CMS + Yoda1 + si-Ctrl group, CMS + Yoda1 + si-CAPN1 group, and CMS + Yoda1 + si-CAPN2 group ( n = 3). ** P < 0. 01, *** P < 0.001, **** P < 0.0001

Article Snippet: Primary antibodies contain calpain1 (10,538–1-AP, Proteintech), calpain2 (11,472–1-AP, Proteintech), Bax (50,599–2-Ig, Proteintech), cleaved-Caspase 3 (9664, Cell Signaling Technology, MA, USA), Piezo1 (5939–1-AP, Proteintech), and Gapdh (60,004–1-Ig, Proteintech).

Techniques: Activity Assay, Western Blot, Expressing, Flow Cytometry, TUNEL Assay, Staining, Fluorescence

Journal: Journal of neuropathology and experimental neurology

Article Title: MicroRNA Alteration in Developing Rat Oligodendrocyte Precursor Cells Induced by Hypoxia-Ischemia.

doi: 10.1093/jnen/nlz071

Figure Lengend Snippet: FIGURE 4. Capn6 expression detected by double immunofluorescence staining and Western blotting. The expression of Capn6 in OPCs was detected by double immunofluorescence staining and Western blotting. (A) O4 (green) and Capn6 (red) double immunofluorescence staining showing Capn6 expression in the region of the CC. Scale bar: 20 mm. Arrows indicate Capn6þ/ O4þ merged cells. Data were presented as means 6 SD (n ¼ 6). Quantitative analysis showed significant difference in the number of O4þ cells and Capn6þ/O4þ merged cells in the CC between sham and HI group. The result indicated that the HI group had a significantly lower expression of Capn6 in the brain of rats compared with the sham. (B) Capn6 expression on postnatal day 15 in HI and sham brains was detected via Western blotting. b-actin was used as the loading control. Values were expressed as relative optical density and represented as mean 6 SD (n ¼ 6). It showed that Capn6 expression was significantly decreased in HI brains compared with sham. *p < 0.05 and **p < 0.01 compared with the sham. CC, corpus callosum; HI, hypoxia-ischemia; OPCs, oligodendrocyte precursor cells; O4, OPC marker.

Article Snippet: The membranes were incubated overnight at 4 C with the following antibodies: rabbit anti-Capn6 monoclonal antibody (Proteintech, 1:200), mouse antiactin polyclonal antibody (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA) was detected as the loading control.

Techniques: Expressing, Double Immunofluorescence Staining, Western Blot, Control, Marker