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mouse anti human clca1 antibody 8d3  (Santa Cruz Biotechnology)


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    Santa Cruz Biotechnology mouse anti human clca1 antibody 8d3
    CLCA4 self-cleavage is required to potentiate I CaCC in HEK293T cells. A , domain schematic representation of human CLCA4 constructs used in this study. The <t>anti-CLCA1</t> antibody <t>8D3</t> is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D , CLCA4 is cleaved and secreted. B , Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C , Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D , Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E , anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F , CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e. , I CaCC . Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G , current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).
    Mouse Anti Human Clca1 Antibody 8d3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+human+clca1+antibody+8d3/CLCA1+Antibody/pmc11231702-96-0-17
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    mouse anti human clca1 antibody 8d3 - by Bioz Stars, 2026-11
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    1) Product Images from "Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells"

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2024.107432

    CLCA4 self-cleavage is required to potentiate I CaCC in HEK293T cells. A , domain schematic representation of human CLCA4 constructs used in this study. The anti-CLCA1 antibody 8D3 is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D , CLCA4 is cleaved and secreted. B , Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C , Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D , Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E , anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F , CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e. , I CaCC . Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G , current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).
    Figure Legend Snippet: CLCA4 self-cleavage is required to potentiate I CaCC in HEK293T cells. A , domain schematic representation of human CLCA4 constructs used in this study. The anti-CLCA1 antibody 8D3 is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D , CLCA4 is cleaved and secreted. B , Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C , Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D , Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E , anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F , CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e. , I CaCC . Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G , current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).

    Techniques Used: Construct, Sequencing, Western Blot, Transfection, Expressing, Plasmid Preparation, Patch Clamp

    CLCA4-dependent I CaCC are carried by TMEM16B, and paracrine CLCA4-TMEM16B interactions are mediated by a MIDAS motif in the CLCA4 VWA domain. A , HEK293T cells transfected with scramble (siControl), TMEM16A siRNA, or TMEM16B siRNA were cultured in medium from mock-transfected cells or from cells expressing full-length CLCA4 or CLCA4 VWA domain (VWA), and assayed by whole-cell patch clamp. B and C , I CaCC were activated in CLCA4- ( B ) and VWA-conditioned ( C ) cells transfected with siControl and TMEM16A siRNA, but current density remained at background levels in cells transfected with TMEM16B siRNA. D , homology model of the CLCA4 VWA domain, based on the crystal structure of CLCA1 VWA (PDB ID: 6PYO ) . Residues composing the MIDAS site and the N-linked glycosylation site (N340) are shown as sticks. E , purified CLCA4 VWA expressed in the presence of kifunensine was treated with the deglycosylation enzyme EndoHf, resulting in a shift to lower molecular weight. F , mutation of the CLCA4 VWA glycosylation site (N340Q) results in a single band corresponding to the non-glycosylated protein. G , CLCA4-dependent I CaCC potentiation was reduced in naïve HEK293T cells conditioned with MIDAS motif double mutant S316A/T383A VWA. Experiments were performed as in ( A ). B , C and G , current density at +100 mV, measured at the end of the 600-ms voltage pulse (protocol as in <xref ref-type=Fig. 1 F ). Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences were evaluated by Kruskal-Wallis one-way ANOVA on ranks, as follows: ( B ) H (5) = 26.647, p < 0.001; ( C ) H (3) = 15.780, p = 0.001; and ( G ) H (2) = 9.267, p = 0.010. Post hoc all-pairwise comparisons between groups were determined by Dunn’s method. Statistical differences are indicated in ( B ) between mock- and CLCA4-conditioned media for each siRNA transfection in CLCA4-conditioned versus scrambled siRNA control in mock-conditioned. Statistical differences are indicated in ( C ) between mock- and CLCA4-conditioned media for each siRNA transfection. Statistical differences are indicated in ( G ) between all treatments. " title="... VWA domain, based on the crystal structure of CLCA1 VWA (PDB ID: 6PYO ) . Residues composing ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: CLCA4-dependent I CaCC are carried by TMEM16B, and paracrine CLCA4-TMEM16B interactions are mediated by a MIDAS motif in the CLCA4 VWA domain. A , HEK293T cells transfected with scramble (siControl), TMEM16A siRNA, or TMEM16B siRNA were cultured in medium from mock-transfected cells or from cells expressing full-length CLCA4 or CLCA4 VWA domain (VWA), and assayed by whole-cell patch clamp. B and C , I CaCC were activated in CLCA4- ( B ) and VWA-conditioned ( C ) cells transfected with siControl and TMEM16A siRNA, but current density remained at background levels in cells transfected with TMEM16B siRNA. D , homology model of the CLCA4 VWA domain, based on the crystal structure of CLCA1 VWA (PDB ID: 6PYO ) . Residues composing the MIDAS site and the N-linked glycosylation site (N340) are shown as sticks. E , purified CLCA4 VWA expressed in the presence of kifunensine was treated with the deglycosylation enzyme EndoHf, resulting in a shift to lower molecular weight. F , mutation of the CLCA4 VWA glycosylation site (N340Q) results in a single band corresponding to the non-glycosylated protein. G , CLCA4-dependent I CaCC potentiation was reduced in naïve HEK293T cells conditioned with MIDAS motif double mutant S316A/T383A VWA. Experiments were performed as in ( A ). B , C and G , current density at +100 mV, measured at the end of the 600-ms voltage pulse (protocol as in Fig. 1 F ). Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences were evaluated by Kruskal-Wallis one-way ANOVA on ranks, as follows: ( B ) H (5) = 26.647, p < 0.001; ( C ) H (3) = 15.780, p = 0.001; and ( G ) H (2) = 9.267, p = 0.010. Post hoc all-pairwise comparisons between groups were determined by Dunn’s method. Statistical differences are indicated in ( B ) between mock- and CLCA4-conditioned media for each siRNA transfection in CLCA4-conditioned versus scrambled siRNA control in mock-conditioned. Statistical differences are indicated in ( C ) between mock- and CLCA4-conditioned media for each siRNA transfection. Statistical differences are indicated in ( G ) between all treatments.

    Techniques Used: Transfection, Cell Culture, Expressing, Patch Clamp, Glycoproteomics, Purification, Molecular Weight, Mutagenesis, Control

    Working model for CLCA family self-activation and engagement of TMEM16 family proteins. A , soluble CLCA1 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA1 and C-CLCA1 fragments. N-CLCA1 engages TMEM16A α9-α10 loop using VWA MIDAS motif. B , membrane-anchored CLCA4 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA4 and C-CLCA4 fragments. N-CLCA4 engages TMEM16B using its VWA domain.
    Figure Legend Snippet: Working model for CLCA family self-activation and engagement of TMEM16 family proteins. A , soluble CLCA1 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA1 and C-CLCA1 fragments. N-CLCA1 engages TMEM16A α9-α10 loop using VWA MIDAS motif. B , membrane-anchored CLCA4 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA4 and C-CLCA4 fragments. N-CLCA4 engages TMEM16B using its VWA domain.

    Techniques Used: Activation Assay, Membrane

    Related Articles

    Construct:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Sequencing:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Western Blot:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Transfection:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Expressing:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Plasmid Preparation:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Patch Clamp:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Cell Culture:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Glycoproteomics:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Purification:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Molecular Weight:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Mutagenesis:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Control:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Activation Assay:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.

    Membrane:

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells
    Article Snippet: General methods were as described ( ).General methods were as described ( ).. Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.. Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.Rabbit anti-6-His-antibody-HRP conjugate (1:10,000; Bethyl Laboratories) was used to detect VWA proteins.



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    mouse anti human clca1 antibody 8d3  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology mouse anti human clca1 antibody 8d3
    CLCA4 self-cleavage is required to potentiate I CaCC in HEK293T cells. A , domain schematic representation of human CLCA4 constructs used in this study. The <t>anti-CLCA1</t> antibody <t>8D3</t> is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D , CLCA4 is cleaved and secreted. B , Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C , Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D , Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E , anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F , CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e. , I CaCC . Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G , current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).
    Mouse Anti Human Clca1 Antibody 8d3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+human+clca1+antibody+8d3/CLCA1+Antibody/pmc11231702-96-0-17
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    CLCA4 self-cleavage is required to potentiate I CaCC in HEK293T cells. A , domain schematic representation of human CLCA4 constructs used in this study. The anti-CLCA1 antibody 8D3 is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D , CLCA4 is cleaved and secreted. B , Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C , Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D , Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E , anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F , CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e. , I CaCC . Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G , current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).

    Journal: The Journal of Biological Chemistry

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

    doi: 10.1016/j.jbc.2024.107432

    Figure Lengend Snippet: CLCA4 self-cleavage is required to potentiate I CaCC in HEK293T cells. A , domain schematic representation of human CLCA4 constructs used in this study. The anti-CLCA1 antibody 8D3 is cross-reactive with CLCA4 and the region containing the epitope is highlighted. Domain abbreviations are as follows: CAT = MMP-like catalytic domain; SS = signal sequence; CYS = MMP-like cysteine-rich domain; VWA = von-Willebrand Type A domain; FnIII = fibronectin type-III domain; GPI = glycosylphosphatidylinositol anchor. B–D , CLCA4 is cleaved and secreted. B , Western blot (using 8D3) of cells transfected with CLCA4. Full-length CLCA4 is only detected in solubilized cells, whereas the cleaved N-terminal fragment (N-CLCA4) is found in both cell lysate and secreted into the medium. C , Western blot (using anti-FLAG M2) of cells transfected with CLCA4-FLAG. Both full-length and C-CLCA4 are only detected in solubilized cells. D , Western blot (using 8D3) of cells transfected with either WT CLCA4 or CLCA4 containing mutations to predicted active-site residues in CLCA4 MMP-like domain. These mutations (H155A and E156Q) prevent self-cleavage. E , anti-FLAG Western blot of cells transfected with CLCA-4-FLAG treated with PI-PLC to release GPI-anchored proteins. C-CLCA4-FLAG is released into media following PI-PLC treatment. F , CLCA4 self-cleavage is required to potentiate CaCC-like currents, i.e. , I CaCC . Cells were transfected with expression constructs or empty pHLsec plasmid (mock) and assayed by whole-cell patch clamp 48 h later. Representative whole-cell currents; pulse protocol on top. G , current density at +100 mV, measured at the end of the 600-ms voltage pulse. Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences between CLCA4 transfected and other conditions are indicated (Kruskal-Wallis one-way ANOVA on ranks, H (3) = 28.813, p < 0.001, followed by Dunn’s test).

    Article Snippet: Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.

    Techniques: Construct, Sequencing, Western Blot, Transfection, Expressing, Plasmid Preparation, Patch Clamp

    CLCA4-dependent I CaCC are carried by TMEM16B, and paracrine CLCA4-TMEM16B interactions are mediated by a MIDAS motif in the CLCA4 VWA domain. A , HEK293T cells transfected with scramble (siControl), TMEM16A siRNA, or TMEM16B siRNA were cultured in medium from mock-transfected cells or from cells expressing full-length CLCA4 or CLCA4 VWA domain (VWA), and assayed by whole-cell patch clamp. B and C , I CaCC were activated in CLCA4- ( B ) and VWA-conditioned ( C ) cells transfected with siControl and TMEM16A siRNA, but current density remained at background levels in cells transfected with TMEM16B siRNA. D , homology model of the CLCA4 VWA domain, based on the crystal structure of CLCA1 VWA (PDB ID: 6PYO ) . Residues composing the MIDAS site and the N-linked glycosylation site (N340) are shown as sticks. E , purified CLCA4 VWA expressed in the presence of kifunensine was treated with the deglycosylation enzyme EndoHf, resulting in a shift to lower molecular weight. F , mutation of the CLCA4 VWA glycosylation site (N340Q) results in a single band corresponding to the non-glycosylated protein. G , CLCA4-dependent I CaCC potentiation was reduced in naïve HEK293T cells conditioned with MIDAS motif double mutant S316A/T383A VWA. Experiments were performed as in ( A ). B , C and G , current density at +100 mV, measured at the end of the 600-ms voltage pulse (protocol as in <xref ref-type=Fig. 1 F ). Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences were evaluated by Kruskal-Wallis one-way ANOVA on ranks, as follows: ( B ) H (5) = 26.647, p < 0.001; ( C ) H (3) = 15.780, p = 0.001; and ( G ) H (2) = 9.267, p = 0.010. Post hoc all-pairwise comparisons between groups were determined by Dunn’s method. Statistical differences are indicated in ( B ) between mock- and CLCA4-conditioned media for each siRNA transfection in CLCA4-conditioned versus scrambled siRNA control in mock-conditioned. Statistical differences are indicated in ( C ) between mock- and CLCA4-conditioned media for each siRNA transfection. Statistical differences are indicated in ( G ) between all treatments. " width="100%" height="100%">

    Journal: The Journal of Biological Chemistry

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

    doi: 10.1016/j.jbc.2024.107432

    Figure Lengend Snippet: CLCA4-dependent I CaCC are carried by TMEM16B, and paracrine CLCA4-TMEM16B interactions are mediated by a MIDAS motif in the CLCA4 VWA domain. A , HEK293T cells transfected with scramble (siControl), TMEM16A siRNA, or TMEM16B siRNA were cultured in medium from mock-transfected cells or from cells expressing full-length CLCA4 or CLCA4 VWA domain (VWA), and assayed by whole-cell patch clamp. B and C , I CaCC were activated in CLCA4- ( B ) and VWA-conditioned ( C ) cells transfected with siControl and TMEM16A siRNA, but current density remained at background levels in cells transfected with TMEM16B siRNA. D , homology model of the CLCA4 VWA domain, based on the crystal structure of CLCA1 VWA (PDB ID: 6PYO ) . Residues composing the MIDAS site and the N-linked glycosylation site (N340) are shown as sticks. E , purified CLCA4 VWA expressed in the presence of kifunensine was treated with the deglycosylation enzyme EndoHf, resulting in a shift to lower molecular weight. F , mutation of the CLCA4 VWA glycosylation site (N340Q) results in a single band corresponding to the non-glycosylated protein. G , CLCA4-dependent I CaCC potentiation was reduced in naïve HEK293T cells conditioned with MIDAS motif double mutant S316A/T383A VWA. Experiments were performed as in ( A ). B , C and G , current density at +100 mV, measured at the end of the 600-ms voltage pulse (protocol as in Fig. 1 F ). Symbols are data from individual patches ( n = 10–25); bars are means ± S.E.M. of all experiments. Statistical differences were evaluated by Kruskal-Wallis one-way ANOVA on ranks, as follows: ( B ) H (5) = 26.647, p < 0.001; ( C ) H (3) = 15.780, p = 0.001; and ( G ) H (2) = 9.267, p = 0.010. Post hoc all-pairwise comparisons between groups were determined by Dunn’s method. Statistical differences are indicated in ( B ) between mock- and CLCA4-conditioned media for each siRNA transfection in CLCA4-conditioned versus scrambled siRNA control in mock-conditioned. Statistical differences are indicated in ( C ) between mock- and CLCA4-conditioned media for each siRNA transfection. Statistical differences are indicated in ( G ) between all treatments.

    Article Snippet: Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.

    Techniques: Transfection, Cell Culture, Expressing, Patch Clamp, Glycoproteomics, Purification, Molecular Weight, Mutagenesis, Control

    Working model for CLCA family self-activation and engagement of TMEM16 family proteins. A , soluble CLCA1 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA1 and C-CLCA1 fragments. N-CLCA1 engages TMEM16A α9-α10 loop using VWA MIDAS motif. B , membrane-anchored CLCA4 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA4 and C-CLCA4 fragments. N-CLCA4 engages TMEM16B using its VWA domain.

    Journal: The Journal of Biological Chemistry

    Article Title: Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells

    doi: 10.1016/j.jbc.2024.107432

    Figure Lengend Snippet: Working model for CLCA family self-activation and engagement of TMEM16 family proteins. A , soluble CLCA1 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA1 and C-CLCA1 fragments. N-CLCA1 engages TMEM16A α9-α10 loop using VWA MIDAS motif. B , membrane-anchored CLCA4 undergoes self-cleavage mediated by its MMP-like domain (CAT) to produce the N-CLCA4 and C-CLCA4 fragments. N-CLCA4 engages TMEM16B using its VWA domain.

    Article Snippet: Mouse anti-human CLCA1 antibody 8D3 developed in-house (1:1000; ( )) and goat anti-mouse IgG antibody-HRP conjugate (1:10,000; Santa Cruz Biotechnology) were used to detect N-terminal CLCA4; and monoclonal anti-FLAG M2-Peroxidase (HRP) antibody (1:5000; MilliporeSigma, Saint Louis, MO) was used to detect C-terminal CLCA4.

    Techniques: Activation Assay, Membrane