anti aquaporin 5 antibody  (Cell Signaling Technology Inc)


Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc anti aquaporin 5 antibody
    LAMP3 increases degradation of NKCC1 and <t>AQP5</t> by promoting endolysosomal degradation in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells) and 48 h thereafter treated with chloroquine (CQ) at 25 μM for 6 h. ( A ) Representative immunofluorescent images for LAMP3 (gray) or NKCC1 (red) and EEA1 (green) (scale bars = 5 µm). ( B – D ) Bar charts showing ( B ) relative change in EEA1 puncta area size, ( C ) Pearson correlation coefficients between EEA1 and NKCC1, and ( D ) relative change in mean fluorescent intensity (MFI) of NKCC1 staining ( n = 3 for all experiments). ( E ) Representative cropped Western blots showing expression of NKCC1, AQP5, and α-tubulin (internal control). Graph shows NKCC1 and AQP5 expression relative to empty ( n = 3). Values shown are mean ± SEM. ** P
    Anti Aquaporin 5 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti aquaporin 5 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti aquaporin 5 antibody - by Bioz Stars, 2022-12
    94/100 stars

    Images

    1) Product Images from "Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy"

    Article Title: Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-21374-2

    LAMP3 increases degradation of NKCC1 and AQP5 by promoting endolysosomal degradation in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells) and 48 h thereafter treated with chloroquine (CQ) at 25 μM for 6 h. ( A ) Representative immunofluorescent images for LAMP3 (gray) or NKCC1 (red) and EEA1 (green) (scale bars = 5 µm). ( B – D ) Bar charts showing ( B ) relative change in EEA1 puncta area size, ( C ) Pearson correlation coefficients between EEA1 and NKCC1, and ( D ) relative change in mean fluorescent intensity (MFI) of NKCC1 staining ( n = 3 for all experiments). ( E ) Representative cropped Western blots showing expression of NKCC1, AQP5, and α-tubulin (internal control). Graph shows NKCC1 and AQP5 expression relative to empty ( n = 3). Values shown are mean ± SEM. ** P
    Figure Legend Snippet: LAMP3 increases degradation of NKCC1 and AQP5 by promoting endolysosomal degradation in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells) and 48 h thereafter treated with chloroquine (CQ) at 25 μM for 6 h. ( A ) Representative immunofluorescent images for LAMP3 (gray) or NKCC1 (red) and EEA1 (green) (scale bars = 5 µm). ( B – D ) Bar charts showing ( B ) relative change in EEA1 puncta area size, ( C ) Pearson correlation coefficients between EEA1 and NKCC1, and ( D ) relative change in mean fluorescent intensity (MFI) of NKCC1 staining ( n = 3 for all experiments). ( E ) Representative cropped Western blots showing expression of NKCC1, AQP5, and α-tubulin (internal control). Graph shows NKCC1 and AQP5 expression relative to empty ( n = 3). Values shown are mean ± SEM. ** P

    Techniques Used: In Vitro, Transfection, Expressing, Plasmid Preparation, Staining, Western Blot

    LAMP3 promotes degradation of NKCC1 and AQP5 in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells). ( A ) Representative cropped Western blots showing expression of NKCC1, AQP5, or α-tubulin (internal control) 48 h after transfection. ( B ) Relative change in SLC12A2 and AQP5 mRNA expressions to ACTB expression determined by real-time quantitative PCR 48 h after transfection. ( C ) Representative cropped Western blots after cells were treated with 50 μg/mL cycloheximide (CHX) for indicated hours. Graph shows remaining protein expression after 8 h relative to baseline. Values shown are mean ± SEM (n = 3 for all experiments). * P
    Figure Legend Snippet: LAMP3 promotes degradation of NKCC1 and AQP5 in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells). ( A ) Representative cropped Western blots showing expression of NKCC1, AQP5, or α-tubulin (internal control) 48 h after transfection. ( B ) Relative change in SLC12A2 and AQP5 mRNA expressions to ACTB expression determined by real-time quantitative PCR 48 h after transfection. ( C ) Representative cropped Western blots after cells were treated with 50 μg/mL cycloheximide (CHX) for indicated hours. Graph shows remaining protein expression after 8 h relative to baseline. Values shown are mean ± SEM (n = 3 for all experiments). * P

    Techniques Used: In Vitro, Transfection, Expressing, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction

    AAV2-AQP1 gene therapy restores salivary flow rate in LAMP3-overexpressing mice. ( A ) LAMP3 overexpression in submandibular glands of C57BL/6 mice was achieved by retrograde cannulation of AAV2-LAMP3. After 7 months, AAV2-AQP1 or AAV2-GFP (control) was delivered to submandibular glands of LAMP3-overexpressing mice by retrograde cannulation. ( B ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min in mice. ( C ) Representative immunofluorescent images of submandibular glands for NKCC1 (green), AQP5 (green), or AQP1 (magenta) (scale bars = 10 µm). ( D ) NKCC1 and AQP5 expression area in glands. ( E ) Serum anti-SSA/Ro antibody levels. ( F ) Size of lymphocyte infiltration area in glands. Values shown are mean ± SD (3 baseline, 5 AAV2-GFP-treated, and 4 AAV2-AQP1-treated mice). ** P
    Figure Legend Snippet: AAV2-AQP1 gene therapy restores salivary flow rate in LAMP3-overexpressing mice. ( A ) LAMP3 overexpression in submandibular glands of C57BL/6 mice was achieved by retrograde cannulation of AAV2-LAMP3. After 7 months, AAV2-AQP1 or AAV2-GFP (control) was delivered to submandibular glands of LAMP3-overexpressing mice by retrograde cannulation. ( B ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min in mice. ( C ) Representative immunofluorescent images of submandibular glands for NKCC1 (green), AQP5 (green), or AQP1 (magenta) (scale bars = 10 µm). ( D ) NKCC1 and AQP5 expression area in glands. ( E ) Serum anti-SSA/Ro antibody levels. ( F ) Size of lymphocyte infiltration area in glands. Values shown are mean ± SD (3 baseline, 5 AAV2-GFP-treated, and 4 AAV2-AQP1-treated mice). ** P

    Techniques Used: Mouse Assay, Over Expression, Expressing

    Graphical summary. LAMP3 overexpression decreases AQP5—a water channel in the apical membrane—and NKCC1—an ion cotransporter on the basolateral membrane—expressions in salivary gland epithelial cells by promoting endolysosomal degradation, resulting in salivary gland hypofunction. Simultaneously, LAMP3 increases internalization of AAV2 and enhances AAV2-mediated gene transduction efficiency via the activated endolysosomal pathway. AAV2-mediated AQP1 gene therapy induces ectopic AQP1 expression in both apical and basolateral membranes of salivary gland epithelial cells and creates a new pathway for fluid movement, leading to restoration of salivary flow rate in LAMP3-overexpressing mice.
    Figure Legend Snippet: Graphical summary. LAMP3 overexpression decreases AQP5—a water channel in the apical membrane—and NKCC1—an ion cotransporter on the basolateral membrane—expressions in salivary gland epithelial cells by promoting endolysosomal degradation, resulting in salivary gland hypofunction. Simultaneously, LAMP3 increases internalization of AAV2 and enhances AAV2-mediated gene transduction efficiency via the activated endolysosomal pathway. AAV2-mediated AQP1 gene therapy induces ectopic AQP1 expression in both apical and basolateral membranes of salivary gland epithelial cells and creates a new pathway for fluid movement, leading to restoration of salivary flow rate in LAMP3-overexpressing mice.

    Techniques Used: Over Expression, Transduction, Expressing, Mouse Assay

    LAMP3 causes salivary gland hypofunction through endolysosomal degradation of NKCC1 and AQP5 in vivo. Submandibular glands of C57BL/6 mice were instilled with AAV2-LAMP3 or AAV2-GFP (control), after which mice received weekly intraperitoneal injections of hydroxychloroquine (HCQ) at 60 mg/kg for 4 months. ( A ) Representative immunofluorescent images of submandibular glands showing staining for NKCC1 or AQP5 (both green) (scale bars = 20 µm). ( B , C ) Bar charts showing percentage of ( B ) NKCC1 and ( C ) AQP5 expressions per nuclear area in submandibular gland specimens. ( D ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min. Values shown are mean ± SD (4 control, 5 LAMP3-overexpressing, and 4 HCQ-treated LAMP3-overexpressing mice). ** P
    Figure Legend Snippet: LAMP3 causes salivary gland hypofunction through endolysosomal degradation of NKCC1 and AQP5 in vivo. Submandibular glands of C57BL/6 mice were instilled with AAV2-LAMP3 or AAV2-GFP (control), after which mice received weekly intraperitoneal injections of hydroxychloroquine (HCQ) at 60 mg/kg for 4 months. ( A ) Representative immunofluorescent images of submandibular glands showing staining for NKCC1 or AQP5 (both green) (scale bars = 20 µm). ( B , C ) Bar charts showing percentage of ( B ) NKCC1 and ( C ) AQP5 expressions per nuclear area in submandibular gland specimens. ( D ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min. Values shown are mean ± SD (4 control, 5 LAMP3-overexpressing, and 4 HCQ-treated LAMP3-overexpressing mice). ** P

    Techniques Used: In Vivo, Mouse Assay, Staining

    Decreased NKCC1 and AQP5 expressions are associated with salivary gland hypofunction in SjD patients. ( A ) Individuals who visited a rheumatology clinic because of sicca symptoms underwent labial minor salivary glands (MSGs) biopsy, measurement of salivary flow rate (SFR) and serum anti-Ro/SSA antibody tests. Thereafter, participants were divided into two groups: SjD patients with decreased SFR ( n = 5) and control subjects with normal SFR ( n = 6). ( B ) Representative immunofluorescent images of MSGs indicating staining for NKCC1 or AQP5 (green) and dot plots showing mean fluorescent intensity (MFI) of NKCC1 or AQP5 staining in each MSG (scale bars = 50 µm). Values shown are mean ± SD. * P
    Figure Legend Snippet: Decreased NKCC1 and AQP5 expressions are associated with salivary gland hypofunction in SjD patients. ( A ) Individuals who visited a rheumatology clinic because of sicca symptoms underwent labial minor salivary glands (MSGs) biopsy, measurement of salivary flow rate (SFR) and serum anti-Ro/SSA antibody tests. Thereafter, participants were divided into two groups: SjD patients with decreased SFR ( n = 5) and control subjects with normal SFR ( n = 6). ( B ) Representative immunofluorescent images of MSGs indicating staining for NKCC1 or AQP5 (green) and dot plots showing mean fluorescent intensity (MFI) of NKCC1 or AQP5 staining in each MSG (scale bars = 50 µm). Values shown are mean ± SD. * P

    Techniques Used: Staining

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Cell Signaling Technology Inc anti aquaporin 5 antibody
    LAMP3 increases degradation of NKCC1 and <t>AQP5</t> by promoting endolysosomal degradation in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells) and 48 h thereafter treated with chloroquine (CQ) at 25 μM for 6 h. ( A ) Representative immunofluorescent images for LAMP3 (gray) or NKCC1 (red) and EEA1 (green) (scale bars = 5 µm). ( B – D ) Bar charts showing ( B ) relative change in EEA1 puncta area size, ( C ) Pearson correlation coefficients between EEA1 and NKCC1, and ( D ) relative change in mean fluorescent intensity (MFI) of NKCC1 staining ( n = 3 for all experiments). ( E ) Representative cropped Western blots showing expression of NKCC1, AQP5, and α-tubulin (internal control). Graph shows NKCC1 and AQP5 expression relative to empty ( n = 3). Values shown are mean ± SEM. ** P
    Anti Aquaporin 5 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti aquaporin 5 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti aquaporin 5 antibody - by Bioz Stars, 2022-12
    94/100 stars
      Buy from Supplier

    94
    Alomone Labs rabbit anti tpcn2 monoclonal antibodies
    Knockdown of <t>TPCN2</t> induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P
    Rabbit Anti Tpcn2 Monoclonal Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti tpcn2 monoclonal antibodies/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti tpcn2 monoclonal antibodies - by Bioz Stars, 2022-12
    94/100 stars
      Buy from Supplier

    95
    Alomone Labs co incubated with antibodies anti nav1 5
    The <t>SCN5A</t> DNA promoter region contains conserved Meis1 binding sites. (A) Conservative Meis1 binding sites in the proximal region (2000 nt upstream) of SCN5A DNA promoter computational predicted by JASPAR database (JASPAR 2020 ). (B) ChIP assay was performed by using <t>antibody</t> against Meis1, and IgG as control on the mice ventricular myocardium in sham or MI group. (C-D) Real-time PCR was performed to evaluate the expression of Meis1 mRNA in neonatal mouse cardiomyocytes by knockdown of Meis1 with si-Meis1 or overexpressed of Meis1 by plasmid. At least six independent batches of cells for each group, ** P
    Co Incubated With Antibodies Anti Nav1 5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/co incubated with antibodies anti nav1 5/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    co incubated with antibodies anti nav1 5 - by Bioz Stars, 2022-12
    95/100 stars
      Buy from Supplier

    94
    Alomone Labs rabbit polyclonal anti trpm4 antibody
    Application of MβCD to the cytoplasmic bath decreases <t>TRPM4</t> channel activity which is reversed by exogenous cholesterol. (A) A representative single channel recording from an inside-out patch shows TRPM4 activity before and after replacement of control cytoplasmic bath solution first with a solution containing 0.5 mM MβCD and then with a solution containing 30 μg/ml cholesterol. “C” indicates channel at the closed state. “O” indicates single-level openings. “I, II, and III” are zoom-ins of the single-channel recording. (B) Summary plots of TRPM4 channel P O under each indicted condition. n = 5 paired experiments, ** P
    Rabbit Polyclonal Anti Trpm4 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti trpm4 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti trpm4 antibody - by Bioz Stars, 2022-12
    94/100 stars
      Buy from Supplier

    Image Search Results


    LAMP3 increases degradation of NKCC1 and AQP5 by promoting endolysosomal degradation in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells) and 48 h thereafter treated with chloroquine (CQ) at 25 μM for 6 h. ( A ) Representative immunofluorescent images for LAMP3 (gray) or NKCC1 (red) and EEA1 (green) (scale bars = 5 µm). ( B – D ) Bar charts showing ( B ) relative change in EEA1 puncta area size, ( C ) Pearson correlation coefficients between EEA1 and NKCC1, and ( D ) relative change in mean fluorescent intensity (MFI) of NKCC1 staining ( n = 3 for all experiments). ( E ) Representative cropped Western blots showing expression of NKCC1, AQP5, and α-tubulin (internal control). Graph shows NKCC1 and AQP5 expression relative to empty ( n = 3). Values shown are mean ± SEM. ** P

    Journal: Scientific Reports

    Article Title: Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

    doi: 10.1038/s41598-022-21374-2

    Figure Lengend Snippet: LAMP3 increases degradation of NKCC1 and AQP5 by promoting endolysosomal degradation in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells) and 48 h thereafter treated with chloroquine (CQ) at 25 μM for 6 h. ( A ) Representative immunofluorescent images for LAMP3 (gray) or NKCC1 (red) and EEA1 (green) (scale bars = 5 µm). ( B – D ) Bar charts showing ( B ) relative change in EEA1 puncta area size, ( C ) Pearson correlation coefficients between EEA1 and NKCC1, and ( D ) relative change in mean fluorescent intensity (MFI) of NKCC1 staining ( n = 3 for all experiments). ( E ) Representative cropped Western blots showing expression of NKCC1, AQP5, and α-tubulin (internal control). Graph shows NKCC1 and AQP5 expression relative to empty ( n = 3). Values shown are mean ± SEM. ** P

    Article Snippet: Membranes were blocked with 2% non-fat dried milk at 25 °C for 1 h and then incubated at 4 °C overnight with one of the following primary antibodies: anti-NKCC1 (#4828, Cell Signaling Technology), anti-AQP5 (#AQP-005, Alomone labs), anti-caveolin-1 (#3267, Cell Signaling Technology), anti-clathrin heavy chain (#4796, Cell Signaling Technology), anti-KIAA0319L/AAVR (#21,016-1-AP, Proteintech), or anti-α-tubulin (#T6199, Sigma-Aldrich).

    Techniques: In Vitro, Transfection, Expressing, Plasmid Preparation, Staining, Western Blot

    LAMP3 promotes degradation of NKCC1 and AQP5 in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells). ( A ) Representative cropped Western blots showing expression of NKCC1, AQP5, or α-tubulin (internal control) 48 h after transfection. ( B ) Relative change in SLC12A2 and AQP5 mRNA expressions to ACTB expression determined by real-time quantitative PCR 48 h after transfection. ( C ) Representative cropped Western blots after cells were treated with 50 μg/mL cycloheximide (CHX) for indicated hours. Graph shows remaining protein expression after 8 h relative to baseline. Values shown are mean ± SEM (n = 3 for all experiments). * P

    Journal: Scientific Reports

    Article Title: Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

    doi: 10.1038/s41598-022-21374-2

    Figure Lengend Snippet: LAMP3 promotes degradation of NKCC1 and AQP5 in vitro. HSG cells were transfected with LAMP3 expression or empty plasmid (3.0 μg per 1 × 10 6 cells). ( A ) Representative cropped Western blots showing expression of NKCC1, AQP5, or α-tubulin (internal control) 48 h after transfection. ( B ) Relative change in SLC12A2 and AQP5 mRNA expressions to ACTB expression determined by real-time quantitative PCR 48 h after transfection. ( C ) Representative cropped Western blots after cells were treated with 50 μg/mL cycloheximide (CHX) for indicated hours. Graph shows remaining protein expression after 8 h relative to baseline. Values shown are mean ± SEM (n = 3 for all experiments). * P

    Article Snippet: Membranes were blocked with 2% non-fat dried milk at 25 °C for 1 h and then incubated at 4 °C overnight with one of the following primary antibodies: anti-NKCC1 (#4828, Cell Signaling Technology), anti-AQP5 (#AQP-005, Alomone labs), anti-caveolin-1 (#3267, Cell Signaling Technology), anti-clathrin heavy chain (#4796, Cell Signaling Technology), anti-KIAA0319L/AAVR (#21,016-1-AP, Proteintech), or anti-α-tubulin (#T6199, Sigma-Aldrich).

    Techniques: In Vitro, Transfection, Expressing, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction

    AAV2-AQP1 gene therapy restores salivary flow rate in LAMP3-overexpressing mice. ( A ) LAMP3 overexpression in submandibular glands of C57BL/6 mice was achieved by retrograde cannulation of AAV2-LAMP3. After 7 months, AAV2-AQP1 or AAV2-GFP (control) was delivered to submandibular glands of LAMP3-overexpressing mice by retrograde cannulation. ( B ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min in mice. ( C ) Representative immunofluorescent images of submandibular glands for NKCC1 (green), AQP5 (green), or AQP1 (magenta) (scale bars = 10 µm). ( D ) NKCC1 and AQP5 expression area in glands. ( E ) Serum anti-SSA/Ro antibody levels. ( F ) Size of lymphocyte infiltration area in glands. Values shown are mean ± SD (3 baseline, 5 AAV2-GFP-treated, and 4 AAV2-AQP1-treated mice). ** P

    Journal: Scientific Reports

    Article Title: Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

    doi: 10.1038/s41598-022-21374-2

    Figure Lengend Snippet: AAV2-AQP1 gene therapy restores salivary flow rate in LAMP3-overexpressing mice. ( A ) LAMP3 overexpression in submandibular glands of C57BL/6 mice was achieved by retrograde cannulation of AAV2-LAMP3. After 7 months, AAV2-AQP1 or AAV2-GFP (control) was delivered to submandibular glands of LAMP3-overexpressing mice by retrograde cannulation. ( B ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min in mice. ( C ) Representative immunofluorescent images of submandibular glands for NKCC1 (green), AQP5 (green), or AQP1 (magenta) (scale bars = 10 µm). ( D ) NKCC1 and AQP5 expression area in glands. ( E ) Serum anti-SSA/Ro antibody levels. ( F ) Size of lymphocyte infiltration area in glands. Values shown are mean ± SD (3 baseline, 5 AAV2-GFP-treated, and 4 AAV2-AQP1-treated mice). ** P

    Article Snippet: Membranes were blocked with 2% non-fat dried milk at 25 °C for 1 h and then incubated at 4 °C overnight with one of the following primary antibodies: anti-NKCC1 (#4828, Cell Signaling Technology), anti-AQP5 (#AQP-005, Alomone labs), anti-caveolin-1 (#3267, Cell Signaling Technology), anti-clathrin heavy chain (#4796, Cell Signaling Technology), anti-KIAA0319L/AAVR (#21,016-1-AP, Proteintech), or anti-α-tubulin (#T6199, Sigma-Aldrich).

    Techniques: Mouse Assay, Over Expression, Expressing

    Graphical summary. LAMP3 overexpression decreases AQP5—a water channel in the apical membrane—and NKCC1—an ion cotransporter on the basolateral membrane—expressions in salivary gland epithelial cells by promoting endolysosomal degradation, resulting in salivary gland hypofunction. Simultaneously, LAMP3 increases internalization of AAV2 and enhances AAV2-mediated gene transduction efficiency via the activated endolysosomal pathway. AAV2-mediated AQP1 gene therapy induces ectopic AQP1 expression in both apical and basolateral membranes of salivary gland epithelial cells and creates a new pathway for fluid movement, leading to restoration of salivary flow rate in LAMP3-overexpressing mice.

    Journal: Scientific Reports

    Article Title: Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

    doi: 10.1038/s41598-022-21374-2

    Figure Lengend Snippet: Graphical summary. LAMP3 overexpression decreases AQP5—a water channel in the apical membrane—and NKCC1—an ion cotransporter on the basolateral membrane—expressions in salivary gland epithelial cells by promoting endolysosomal degradation, resulting in salivary gland hypofunction. Simultaneously, LAMP3 increases internalization of AAV2 and enhances AAV2-mediated gene transduction efficiency via the activated endolysosomal pathway. AAV2-mediated AQP1 gene therapy induces ectopic AQP1 expression in both apical and basolateral membranes of salivary gland epithelial cells and creates a new pathway for fluid movement, leading to restoration of salivary flow rate in LAMP3-overexpressing mice.

    Article Snippet: Membranes were blocked with 2% non-fat dried milk at 25 °C for 1 h and then incubated at 4 °C overnight with one of the following primary antibodies: anti-NKCC1 (#4828, Cell Signaling Technology), anti-AQP5 (#AQP-005, Alomone labs), anti-caveolin-1 (#3267, Cell Signaling Technology), anti-clathrin heavy chain (#4796, Cell Signaling Technology), anti-KIAA0319L/AAVR (#21,016-1-AP, Proteintech), or anti-α-tubulin (#T6199, Sigma-Aldrich).

    Techniques: Over Expression, Transduction, Expressing, Mouse Assay

    LAMP3 causes salivary gland hypofunction through endolysosomal degradation of NKCC1 and AQP5 in vivo. Submandibular glands of C57BL/6 mice were instilled with AAV2-LAMP3 or AAV2-GFP (control), after which mice received weekly intraperitoneal injections of hydroxychloroquine (HCQ) at 60 mg/kg for 4 months. ( A ) Representative immunofluorescent images of submandibular glands showing staining for NKCC1 or AQP5 (both green) (scale bars = 20 µm). ( B , C ) Bar charts showing percentage of ( B ) NKCC1 and ( C ) AQP5 expressions per nuclear area in submandibular gland specimens. ( D ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min. Values shown are mean ± SD (4 control, 5 LAMP3-overexpressing, and 4 HCQ-treated LAMP3-overexpressing mice). ** P

    Journal: Scientific Reports

    Article Title: Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

    doi: 10.1038/s41598-022-21374-2

    Figure Lengend Snippet: LAMP3 causes salivary gland hypofunction through endolysosomal degradation of NKCC1 and AQP5 in vivo. Submandibular glands of C57BL/6 mice were instilled with AAV2-LAMP3 or AAV2-GFP (control), after which mice received weekly intraperitoneal injections of hydroxychloroquine (HCQ) at 60 mg/kg for 4 months. ( A ) Representative immunofluorescent images of submandibular glands showing staining for NKCC1 or AQP5 (both green) (scale bars = 20 µm). ( B , C ) Bar charts showing percentage of ( B ) NKCC1 and ( C ) AQP5 expressions per nuclear area in submandibular gland specimens. ( D ) Pilocarpine-stimulated salivary flow rate per body weight in 20 min. Values shown are mean ± SD (4 control, 5 LAMP3-overexpressing, and 4 HCQ-treated LAMP3-overexpressing mice). ** P

    Article Snippet: Membranes were blocked with 2% non-fat dried milk at 25 °C for 1 h and then incubated at 4 °C overnight with one of the following primary antibodies: anti-NKCC1 (#4828, Cell Signaling Technology), anti-AQP5 (#AQP-005, Alomone labs), anti-caveolin-1 (#3267, Cell Signaling Technology), anti-clathrin heavy chain (#4796, Cell Signaling Technology), anti-KIAA0319L/AAVR (#21,016-1-AP, Proteintech), or anti-α-tubulin (#T6199, Sigma-Aldrich).

    Techniques: In Vivo, Mouse Assay, Staining

    Decreased NKCC1 and AQP5 expressions are associated with salivary gland hypofunction in SjD patients. ( A ) Individuals who visited a rheumatology clinic because of sicca symptoms underwent labial minor salivary glands (MSGs) biopsy, measurement of salivary flow rate (SFR) and serum anti-Ro/SSA antibody tests. Thereafter, participants were divided into two groups: SjD patients with decreased SFR ( n = 5) and control subjects with normal SFR ( n = 6). ( B ) Representative immunofluorescent images of MSGs indicating staining for NKCC1 or AQP5 (green) and dot plots showing mean fluorescent intensity (MFI) of NKCC1 or AQP5 staining in each MSG (scale bars = 50 µm). Values shown are mean ± SD. * P

    Journal: Scientific Reports

    Article Title: Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

    doi: 10.1038/s41598-022-21374-2

    Figure Lengend Snippet: Decreased NKCC1 and AQP5 expressions are associated with salivary gland hypofunction in SjD patients. ( A ) Individuals who visited a rheumatology clinic because of sicca symptoms underwent labial minor salivary glands (MSGs) biopsy, measurement of salivary flow rate (SFR) and serum anti-Ro/SSA antibody tests. Thereafter, participants were divided into two groups: SjD patients with decreased SFR ( n = 5) and control subjects with normal SFR ( n = 6). ( B ) Representative immunofluorescent images of MSGs indicating staining for NKCC1 or AQP5 (green) and dot plots showing mean fluorescent intensity (MFI) of NKCC1 or AQP5 staining in each MSG (scale bars = 50 µm). Values shown are mean ± SD. * P

    Article Snippet: Membranes were blocked with 2% non-fat dried milk at 25 °C for 1 h and then incubated at 4 °C overnight with one of the following primary antibodies: anti-NKCC1 (#4828, Cell Signaling Technology), anti-AQP5 (#AQP-005, Alomone labs), anti-caveolin-1 (#3267, Cell Signaling Technology), anti-clathrin heavy chain (#4796, Cell Signaling Technology), anti-KIAA0319L/AAVR (#21,016-1-AP, Proteintech), or anti-α-tubulin (#T6199, Sigma-Aldrich).

    Techniques: Staining

    Knockdown of TPCN2 induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: Knockdown of TPCN2 induces apoptosis and cell-cycle arrest in SLE. Apoptosis was detected by flow cytometry with Annexin V and PI in jurkat- TPCN2 -knockdown cells (A) and THP-1- TPCN2 -knockdown cells (B). TPCN2 -knockdown induced G2/M cell-cycle arrest. The percentage of G2-M phase cells of Jurkat (C) and THP-1 (D) was assessed by flow cytometry. ∗ P

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: Flow Cytometry

    TPCN2 -knockdown inhibits the proliferation in SLE. (A) Expression level of TPCN2 in PBMCs of SLE cases ( n = 6) and healthy controls ( n = 7). ∗ P

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: TPCN2 -knockdown inhibits the proliferation in SLE. (A) Expression level of TPCN2 in PBMCs of SLE cases ( n = 6) and healthy controls ( n = 7). ∗ P

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: Expressing

    RNA-seq analyses of the effect of TPCN2 -knockdown on the gene expression profile. (A) The heatmap shows differential expression genes in sh#1 and sh#2 (in comparison with NC group). (B) The differential statistics of DEGs in NC groups vs . sh#1/sh#2. Blue columnar represent up-regulated DEGs. Red column represents down-regulated DEGs. (C) GO classification analysis of DEGs. NC group vs . sh#2. Molecular function – blue; Cellular components – red; Biological process – green. (D) Top 20 enriched KEGG pathways after silencing TPCN2 in Jurkat. The x-axis is the enrichment score, and the y-axis is the enriched pathways. DEGs: Differentially expressed genes; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; NC: Normal control; RNA-seq: RNA sequencing; Sh#1: shTPCN2#1; Sh#2: shTPCN2#2; TPCN2 : Two-pore segment channel 2.

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: RNA-seq analyses of the effect of TPCN2 -knockdown on the gene expression profile. (A) The heatmap shows differential expression genes in sh#1 and sh#2 (in comparison with NC group). (B) The differential statistics of DEGs in NC groups vs . sh#1/sh#2. Blue columnar represent up-regulated DEGs. Red column represents down-regulated DEGs. (C) GO classification analysis of DEGs. NC group vs . sh#2. Molecular function – blue; Cellular components – red; Biological process – green. (D) Top 20 enriched KEGG pathways after silencing TPCN2 in Jurkat. The x-axis is the enrichment score, and the y-axis is the enriched pathways. DEGs: Differentially expressed genes; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; NC: Normal control; RNA-seq: RNA sequencing; Sh#1: shTPCN2#1; Sh#2: shTPCN2#2; TPCN2 : Two-pore segment channel 2.

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: RNA Sequencing Assay, Expressing

    Representative enriched pathways in high-risk shTPCN2#2 through GSEA analysis. GSEA results showed that the G2/M checkpoint (A), inflammatory response (B), complement (C), and PI3K-AKT-mTOR (D) pathways were enriched in the sh#2 expression group. Top panels indicate the enrichment scores for each gene. Bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes. (E) The mRNA expression of some DEGs in Jurkat cells were detected by qRT-PCR. The RNA was extracted from cells knocked down of TPCN2 with two independent shRNA. The results were shown as the mean ± SD from three independent experiments. ∗ P

    Journal: Chinese Medical Journal

    Article Title: Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

    doi: 10.1097/CM9.0000000000001893

    Figure Lengend Snippet: Representative enriched pathways in high-risk shTPCN2#2 through GSEA analysis. GSEA results showed that the G2/M checkpoint (A), inflammatory response (B), complement (C), and PI3K-AKT-mTOR (D) pathways were enriched in the sh#2 expression group. Top panels indicate the enrichment scores for each gene. Bottom panels show the ranking metrics of each gene. Y-axis: ranking metric values; X-axis: ranks for all genes. (E) The mRNA expression of some DEGs in Jurkat cells were detected by qRT-PCR. The RNA was extracted from cells knocked down of TPCN2 with two independent shRNA. The results were shown as the mean ± SD from three independent experiments. ∗ P

    Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h, and then incubated with rabbit anti-TPCN2 monoclonal antibodies (1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C.

    Techniques: Expressing, Quantitative RT-PCR, shRNA

    The SCN5A DNA promoter region contains conserved Meis1 binding sites. (A) Conservative Meis1 binding sites in the proximal region (2000 nt upstream) of SCN5A DNA promoter computational predicted by JASPAR database (JASPAR 2020 ). (B) ChIP assay was performed by using antibody against Meis1, and IgG as control on the mice ventricular myocardium in sham or MI group. (C-D) Real-time PCR was performed to evaluate the expression of Meis1 mRNA in neonatal mouse cardiomyocytes by knockdown of Meis1 with si-Meis1 or overexpressed of Meis1 by plasmid. At least six independent batches of cells for each group, ** P

    Journal: Journal of Advanced Research

    Article Title: Transcription factor Meis1 act as a new regulator of ischemic arrhythmias in mice

    doi: 10.1016/j.jare.2021.11.004

    Figure Lengend Snippet: The SCN5A DNA promoter region contains conserved Meis1 binding sites. (A) Conservative Meis1 binding sites in the proximal region (2000 nt upstream) of SCN5A DNA promoter computational predicted by JASPAR database (JASPAR 2020 ). (B) ChIP assay was performed by using antibody against Meis1, and IgG as control on the mice ventricular myocardium in sham or MI group. (C-D) Real-time PCR was performed to evaluate the expression of Meis1 mRNA in neonatal mouse cardiomyocytes by knockdown of Meis1 with si-Meis1 or overexpressed of Meis1 by plasmid. At least six independent batches of cells for each group, ** P

    Article Snippet: After permeabilized with 0.5% Triton X-100 for 15 min and blocked with goat serum for 1 h, the samples were co-incubated with antibodies anti-Nav1.5 (Alomane laboratory, Israel, Cat#: ASC-005, 1:200) and anti-α-actinin (ab50599, Abcam, Cambridge, UK, 1:100) overnight at 4 °C.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation

    Meis1 inhibits the down-regulation of SCN5A /Na V 1.5 expression in MI hearts and hypoxic cardiomyocytes. (A-B) The mRNA and protein expression of sodium channel subunit SCN5A /Na V 1.5 in the border zone of infracted hearts from Sham, MI + AAV9-NC and MI + AAV9-Meis1 group (n = 6). (C-D) The expression of SCN5A /Na V 1.5 at mRNA and protein level in neonatal mouse cardiomyocytes after treatment with hypoxia for 24 h with or without forced expression of Meis1 (n = 6). (E) Immunofluorescence staining for Na V 1.5 (green), α-actinin (red) proteins in neonatal mouse cardiomyocytes after treatment with hypoxia for 24 h with or without overexpression of Meis1 (left panels) and enlargement of outlined squares (right panels). Scale bar indicates 5 μm/2 μm. One-way ANOVA was used to determine statistical significance in these experiments. Error bars represent mean ± SEM of each group. * P

    Journal: Journal of Advanced Research

    Article Title: Transcription factor Meis1 act as a new regulator of ischemic arrhythmias in mice

    doi: 10.1016/j.jare.2021.11.004

    Figure Lengend Snippet: Meis1 inhibits the down-regulation of SCN5A /Na V 1.5 expression in MI hearts and hypoxic cardiomyocytes. (A-B) The mRNA and protein expression of sodium channel subunit SCN5A /Na V 1.5 in the border zone of infracted hearts from Sham, MI + AAV9-NC and MI + AAV9-Meis1 group (n = 6). (C-D) The expression of SCN5A /Na V 1.5 at mRNA and protein level in neonatal mouse cardiomyocytes after treatment with hypoxia for 24 h with or without forced expression of Meis1 (n = 6). (E) Immunofluorescence staining for Na V 1.5 (green), α-actinin (red) proteins in neonatal mouse cardiomyocytes after treatment with hypoxia for 24 h with or without overexpression of Meis1 (left panels) and enlargement of outlined squares (right panels). Scale bar indicates 5 μm/2 μm. One-way ANOVA was used to determine statistical significance in these experiments. Error bars represent mean ± SEM of each group. * P

    Article Snippet: After permeabilized with 0.5% Triton X-100 for 15 min and blocked with goat serum for 1 h, the samples were co-incubated with antibodies anti-Nav1.5 (Alomane laboratory, Israel, Cat#: ASC-005, 1:200) and anti-α-actinin (ab50599, Abcam, Cambridge, UK, 1:100) overnight at 4 °C.

    Techniques: Expressing, Immunofluorescence, Staining, Over Expression

    Application of MβCD to the cytoplasmic bath decreases TRPM4 channel activity which is reversed by exogenous cholesterol. (A) A representative single channel recording from an inside-out patch shows TRPM4 activity before and after replacement of control cytoplasmic bath solution first with a solution containing 0.5 mM MβCD and then with a solution containing 30 μg/ml cholesterol. “C” indicates channel at the closed state. “O” indicates single-level openings. “I, II, and III” are zoom-ins of the single-channel recording. (B) Summary plots of TRPM4 channel P O under each indicted condition. n = 5 paired experiments, ** P

    Journal: Frontiers in Pharmacology

    Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

    doi: 10.3389/fphar.2021.627875

    Figure Lengend Snippet: Application of MβCD to the cytoplasmic bath decreases TRPM4 channel activity which is reversed by exogenous cholesterol. (A) A representative single channel recording from an inside-out patch shows TRPM4 activity before and after replacement of control cytoplasmic bath solution first with a solution containing 0.5 mM MβCD and then with a solution containing 30 μg/ml cholesterol. “C” indicates channel at the closed state. “O” indicates single-level openings. “I, II, and III” are zoom-ins of the single-channel recording. (B) Summary plots of TRPM4 channel P O under each indicted condition. n = 5 paired experiments, ** P

    Article Snippet: The membranes were then blocked in 5% non-fat dry milk for 1 h, followed by incubation with rabbit polyclonal anti-TRPM4 antibody (1:200 dilution; Alomone Labs; ACC-044) at 4°C for overnight.

    Techniques: Activity Assay

    Treatment of cells with cholesterol increases, but with lovastatin decreases, TRPM4 channel activity by regulating its sensitivity to Ca 2+ in mpkCCD c14 Cells. (A) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 30 μg/ml cholesterol for 48 hrs. From top to bottom: 1 μM, 10 μM, 200 μM, 1 mM, and 5 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (B) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 5 μM lovastatin for 48 hrs. From top to bottom: 10 μM, 200 μM, 1 mM, 5 mM and 10 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (C) The effect of membrane cholesterol on Ca 2+ -dependence of channel opening. Channel P O was plotted as a function of free Ca 2+ concentration in the bath. P o values are shown for patches either with exogenous cholesterol treatment (black line) or lovastatin treatment (red line). n = 4–7 cells for different data points.

    Journal: Frontiers in Pharmacology

    Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

    doi: 10.3389/fphar.2021.627875

    Figure Lengend Snippet: Treatment of cells with cholesterol increases, but with lovastatin decreases, TRPM4 channel activity by regulating its sensitivity to Ca 2+ in mpkCCD c14 Cells. (A) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 30 μg/ml cholesterol for 48 hrs. From top to bottom: 1 μM, 10 μM, 200 μM, 1 mM, and 5 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (B) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 5 μM lovastatin for 48 hrs. From top to bottom: 10 μM, 200 μM, 1 mM, 5 mM and 10 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (C) The effect of membrane cholesterol on Ca 2+ -dependence of channel opening. Channel P O was plotted as a function of free Ca 2+ concentration in the bath. P o values are shown for patches either with exogenous cholesterol treatment (black line) or lovastatin treatment (red line). n = 4–7 cells for different data points.

    Article Snippet: The membranes were then blocked in 5% non-fat dry milk for 1 h, followed by incubation with rabbit polyclonal anti-TRPM4 antibody (1:200 dilution; Alomone Labs; ACC-044) at 4°C for overnight.

    Techniques: Activity Assay, Concentration Assay

    PI(4, 5)P 2 stimulates TRPM4 via a physical interaction. (A) A representative single channel recording shows that diC8-PI(4,5)P 2 significantly increased TRPM4 activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 5 paired experiments. ** P

    Journal: Frontiers in Pharmacology

    Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

    doi: 10.3389/fphar.2021.627875

    Figure Lengend Snippet: PI(4, 5)P 2 stimulates TRPM4 via a physical interaction. (A) A representative single channel recording shows that diC8-PI(4,5)P 2 significantly increased TRPM4 activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 5 paired experiments. ** P

    Article Snippet: The membranes were then blocked in 5% non-fat dry milk for 1 h, followed by incubation with rabbit polyclonal anti-TRPM4 antibody (1:200 dilution; Alomone Labs; ACC-044) at 4°C for overnight.

    Techniques: Activity Assay

    Cholesterol stimulates TRPM4 via a PI(4, 5)P 2 -dependent mechanism. (A) A representative single channel recording shows that treatment of mpkCCD c14 cells with 20 nM wortmannin had no effect on cholesterol-induced TRPM4 channel activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 6 paired experiments, ** P

    Journal: Frontiers in Pharmacology

    Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

    doi: 10.3389/fphar.2021.627875

    Figure Lengend Snippet: Cholesterol stimulates TRPM4 via a PI(4, 5)P 2 -dependent mechanism. (A) A representative single channel recording shows that treatment of mpkCCD c14 cells with 20 nM wortmannin had no effect on cholesterol-induced TRPM4 channel activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 6 paired experiments, ** P

    Article Snippet: The membranes were then blocked in 5% non-fat dry milk for 1 h, followed by incubation with rabbit polyclonal anti-TRPM4 antibody (1:200 dilution; Alomone Labs; ACC-044) at 4°C for overnight.

    Techniques: Activity Assay

    TRPM4 channels are mainly located in lipid rafts. (A) Representative confocal microscopy images indicate that majority of TRPM4 (green) was co-localized with cholera toxin B (red) in the apical membrane; white rectangular box indicate zoomed-in areas shown in the Zoom-in panels. Data represent five individual experiments showing consistent results (B) Majority of TRPM4 was detected in low-density regions in sucrose gradient experiments; Caveolin-1 was used as a control protein that is known to be located in lipid rafts. Data represent three individual experiments showing consistent results.

    Journal: Frontiers in Pharmacology

    Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

    doi: 10.3389/fphar.2021.627875

    Figure Lengend Snippet: TRPM4 channels are mainly located in lipid rafts. (A) Representative confocal microscopy images indicate that majority of TRPM4 (green) was co-localized with cholera toxin B (red) in the apical membrane; white rectangular box indicate zoomed-in areas shown in the Zoom-in panels. Data represent five individual experiments showing consistent results (B) Majority of TRPM4 was detected in low-density regions in sucrose gradient experiments; Caveolin-1 was used as a control protein that is known to be located in lipid rafts. Data represent three individual experiments showing consistent results.

    Article Snippet: The membranes were then blocked in 5% non-fat dry milk for 1 h, followed by incubation with rabbit polyclonal anti-TRPM4 antibody (1:200 dilution; Alomone Labs; ACC-044) at 4°C for overnight.

    Techniques: Confocal Microscopy

    Treatment with cholesterol or lovastatin does not alter expression levels of TRPM4 in mpkCCDc14 Cells. (A) Representative confocal microscopy images of mpkCCDc14 cells stained with TRPM4 antibody under each indicated conditions. (B) Summary plots of fluorescence intensity of TRPM4. Data are from 24 cells in four sets of separate experiments. (C) Representative Western blots from cell-surface biotinylated and the total proteins of TRPM4 protein. (D) Summary plots of relative expression of TRPM4. Cells were either under control conditions or treated with 30 μg/ml cholesterol alone, 30 μg/ml cholesterol plus 5 μM lovastatin, or 5 μM lovastatin alone for 48 hrs, respectively. n = 5. (E) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing 5 mM CaCl 2 , followed by a bath solution with 10 mM EGTA and no calcium. Cells were either under control conditions or treated with 30 μg/ml cholesterol, or 5 μM lovastatin for 48 hrs, respectively. (F) Summary plots of the number of active channels in the patches under each indicated condition. n = 5 for control cells, n = 5 for cells treated with cholesterol, n = 4 for cells treated with lovastatin.

    Journal: Frontiers in Pharmacology

    Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

    doi: 10.3389/fphar.2021.627875

    Figure Lengend Snippet: Treatment with cholesterol or lovastatin does not alter expression levels of TRPM4 in mpkCCDc14 Cells. (A) Representative confocal microscopy images of mpkCCDc14 cells stained with TRPM4 antibody under each indicated conditions. (B) Summary plots of fluorescence intensity of TRPM4. Data are from 24 cells in four sets of separate experiments. (C) Representative Western blots from cell-surface biotinylated and the total proteins of TRPM4 protein. (D) Summary plots of relative expression of TRPM4. Cells were either under control conditions or treated with 30 μg/ml cholesterol alone, 30 μg/ml cholesterol plus 5 μM lovastatin, or 5 μM lovastatin alone for 48 hrs, respectively. n = 5. (E) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing 5 mM CaCl 2 , followed by a bath solution with 10 mM EGTA and no calcium. Cells were either under control conditions or treated with 30 μg/ml cholesterol, or 5 μM lovastatin for 48 hrs, respectively. (F) Summary plots of the number of active channels in the patches under each indicated condition. n = 5 for control cells, n = 5 for cells treated with cholesterol, n = 4 for cells treated with lovastatin.

    Article Snippet: The membranes were then blocked in 5% non-fat dry milk for 1 h, followed by incubation with rabbit polyclonal anti-TRPM4 antibody (1:200 dilution; Alomone Labs; ACC-044) at 4°C for overnight.

    Techniques: Expressing, Confocal Microscopy, Staining, Fluorescence, Western Blot