clic5  (Alomone Labs)


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    Alomone Labs clic5
    Localization of CLIC1, CLIC4 and <t>CLIC5</t> to the endoplasmic reticulum. (I) Isolated p3 neonatal cardiomyocytes were loaded with ER tracker (A, E, I), fixed, permeabilized, labeled with anti-CLIC1 (B), anti-CLIC4 (F), anti-CLIC5 (J) antibodies and further stained with DAPI (C, G, K). D, H and L are merge images showing colocalization of CLIC1 and CLIC4 to the endoplasmic reticulum whereas CLIC5 shows much less colocalization. D′, H′ and L′ are enlarged images of the squared region. (II) Bar graph representing percentage colocalization of CLIC1, CLIC4 and CLIC5 to endoplasmic reticulum ( n =3).
    Clic5, supplied by Alomone Labs, 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/result/clic5/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    clic5 - by Bioz Stars, 2022-08
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    1) Product Images from "Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes"

    Article Title: Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes

    Journal: Data in Brief

    doi: 10.1016/j.dib.2016.03.061

    Localization of CLIC1, CLIC4 and CLIC5 to the endoplasmic reticulum. (I) Isolated p3 neonatal cardiomyocytes were loaded with ER tracker (A, E, I), fixed, permeabilized, labeled with anti-CLIC1 (B), anti-CLIC4 (F), anti-CLIC5 (J) antibodies and further stained with DAPI (C, G, K). D, H and L are merge images showing colocalization of CLIC1 and CLIC4 to the endoplasmic reticulum whereas CLIC5 shows much less colocalization. D′, H′ and L′ are enlarged images of the squared region. (II) Bar graph representing percentage colocalization of CLIC1, CLIC4 and CLIC5 to endoplasmic reticulum ( n =3).
    Figure Legend Snippet: Localization of CLIC1, CLIC4 and CLIC5 to the endoplasmic reticulum. (I) Isolated p3 neonatal cardiomyocytes were loaded with ER tracker (A, E, I), fixed, permeabilized, labeled with anti-CLIC1 (B), anti-CLIC4 (F), anti-CLIC5 (J) antibodies and further stained with DAPI (C, G, K). D, H and L are merge images showing colocalization of CLIC1 and CLIC4 to the endoplasmic reticulum whereas CLIC5 shows much less colocalization. D′, H′ and L′ are enlarged images of the squared region. (II) Bar graph representing percentage colocalization of CLIC1, CLIC4 and CLIC5 to endoplasmic reticulum ( n =3).

    Techniques Used: Isolation, Labeling, Staining

    Specificity of CLIC antibodies using CLIC1, CLIC4 and CLIC5 knock out (KO) mice heart lysates . (I) 50 µg of heart lysates from WT, clic1 −/− , clic4 −/− and clic5 −/− mouse were electrophoresed on 4–20% (w/v) SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-CLIC1, anti-CLIC4 and anti-CLIC5 antibodies. Absence of CLIC1, CLIC4 and CLIC5 specific bands in KO organ samples confirms the specificity of the antibody. Corresponding Ponceau S stained nitrocellulose membrane is shown as a loading control.
    Figure Legend Snippet: Specificity of CLIC antibodies using CLIC1, CLIC4 and CLIC5 knock out (KO) mice heart lysates . (I) 50 µg of heart lysates from WT, clic1 −/− , clic4 −/− and clic5 −/− mouse were electrophoresed on 4–20% (w/v) SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-CLIC1, anti-CLIC4 and anti-CLIC5 antibodies. Absence of CLIC1, CLIC4 and CLIC5 specific bands in KO organ samples confirms the specificity of the antibody. Corresponding Ponceau S stained nitrocellulose membrane is shown as a loading control.

    Techniques Used: Knock-Out, Mouse Assay, SDS Page, Staining

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    Alomone Labs anti clic5
    Independent localization of <t>CLIC5</t> and GRXCR2 in hair cells during development. (A) Costaining of cochlear whole mounts from wild-type and Clic5 –/– mice at postnatal day 4 (P4) with CLIC5-antibody (green) and phalloidin (red) to reveal stereocilia. Note, no immunostaining signal in Clic5 –/– hair cells, suggesting that the CLIC5-antibody is specific. (B) Costaining of cochlear whole mounts from wild-type and Grxcr2 –/– mice with GRXCR2-antibody and phalloidin. Note, no immunostaining signal in Grxcr2 –/– hair cells, suggesting that the GRXCR2-antibody is specific. (C) Co-staining of P4 cochlear whole mounts from wild-type and Grxcr2 –/– mice with CLIC5-antibody and phalloidin. Note, CLIC5 was concentrated at the base of stereocilia in outer hair cells (OHCs) and inner hair cell (IHCs) of wild-type and Grxcr2 –/– mice. (D) Co-staining of P5 cochlear whole mounts from wild-type and Clic5 –/– mice with GRXCR2-antibody and phalloidin. Note, GRXCR2 was concentrated at the base of stereocilia in wild-type and Clic5 –/– hair cells. Scale bars: 5 μm.
    Anti Clic5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Independent localization of CLIC5 and GRXCR2 in hair cells during development. (A) Costaining of cochlear whole mounts from wild-type and Clic5 –/– mice at postnatal day 4 (P4) with CLIC5-antibody (green) and phalloidin (red) to reveal stereocilia. Note, no immunostaining signal in Clic5 –/– hair cells, suggesting that the CLIC5-antibody is specific. (B) Costaining of cochlear whole mounts from wild-type and Grxcr2 –/– mice with GRXCR2-antibody and phalloidin. Note, no immunostaining signal in Grxcr2 –/– hair cells, suggesting that the GRXCR2-antibody is specific. (C) Co-staining of P4 cochlear whole mounts from wild-type and Grxcr2 –/– mice with CLIC5-antibody and phalloidin. Note, CLIC5 was concentrated at the base of stereocilia in outer hair cells (OHCs) and inner hair cell (IHCs) of wild-type and Grxcr2 –/– mice. (D) Co-staining of P5 cochlear whole mounts from wild-type and Clic5 –/– mice with GRXCR2-antibody and phalloidin. Note, GRXCR2 was concentrated at the base of stereocilia in wild-type and Clic5 –/– hair cells. Scale bars: 5 μm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: N-Terminus of GRXCR2 Interacts With CLIC5 and Is Essential for Auditory Perception

    doi: 10.3389/fcell.2021.671364

    Figure Lengend Snippet: Independent localization of CLIC5 and GRXCR2 in hair cells during development. (A) Costaining of cochlear whole mounts from wild-type and Clic5 –/– mice at postnatal day 4 (P4) with CLIC5-antibody (green) and phalloidin (red) to reveal stereocilia. Note, no immunostaining signal in Clic5 –/– hair cells, suggesting that the CLIC5-antibody is specific. (B) Costaining of cochlear whole mounts from wild-type and Grxcr2 –/– mice with GRXCR2-antibody and phalloidin. Note, no immunostaining signal in Grxcr2 –/– hair cells, suggesting that the GRXCR2-antibody is specific. (C) Co-staining of P4 cochlear whole mounts from wild-type and Grxcr2 –/– mice with CLIC5-antibody and phalloidin. Note, CLIC5 was concentrated at the base of stereocilia in outer hair cells (OHCs) and inner hair cell (IHCs) of wild-type and Grxcr2 –/– mice. (D) Co-staining of P5 cochlear whole mounts from wild-type and Clic5 –/– mice with GRXCR2-antibody and phalloidin. Note, GRXCR2 was concentrated at the base of stereocilia in wild-type and Clic5 –/– hair cells. Scale bars: 5 μm.

    Article Snippet: Primary antibodies were as follows: anti-GRXCR2 (Cat# HPA059421, Sigma); anti-taperin (Cat# HPA020899, Sigma); anti-CLIC5 (Cat# ACL-025, Alomone labs).

    Techniques: Mouse Assay, Immunostaining, Staining

    Glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2) interacts with CLIC5. (A) Diagram of cochlear hair cell showing the staircase-shaped hair bundle. Inside the stereocilia, the tightly cross-linked actin filaments provide the stiffness of stereocilia. GRXCR2 and CLIC5 are concentrated at the base of stereocilia, the site of stereocilia pivoting. (B) Diagram of the constructs used for biochemical experiments. (C–G) HEK293 cells were transfected with the constructs indicated on the top of each panel. Immunoprecipitations were carried out with Myc-antibody (C) or HA-antibody (D–G) followed by western blotting to detect co-expressed proteins. The upper rows show Co-IP results and lower rows show input proteins.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: N-Terminus of GRXCR2 Interacts With CLIC5 and Is Essential for Auditory Perception

    doi: 10.3389/fcell.2021.671364

    Figure Lengend Snippet: Glutaredoxin domain-containing cysteine-rich protein 2 (GRXCR2) interacts with CLIC5. (A) Diagram of cochlear hair cell showing the staircase-shaped hair bundle. Inside the stereocilia, the tightly cross-linked actin filaments provide the stiffness of stereocilia. GRXCR2 and CLIC5 are concentrated at the base of stereocilia, the site of stereocilia pivoting. (B) Diagram of the constructs used for biochemical experiments. (C–G) HEK293 cells were transfected with the constructs indicated on the top of each panel. Immunoprecipitations were carried out with Myc-antibody (C) or HA-antibody (D–G) followed by western blotting to detect co-expressed proteins. The upper rows show Co-IP results and lower rows show input proteins.

    Article Snippet: Primary antibodies were as follows: anti-GRXCR2 (Cat# HPA059421, Sigma); anti-taperin (Cat# HPA020899, Sigma); anti-CLIC5 (Cat# ACL-025, Alomone labs).

    Techniques: Construct, Transfection, Western Blot, Co-Immunoprecipitation Assay

    Disrupting the interaction between GRXCR2 and CLIC5 has minimal effects on stereocilia morphogenesis. (A) Amino acids from 36 to 95 in GRXCR2 are highly conserved in mammals. (B) Diagram of the strategy to generate Grxcr2 D 180/D180 mice. sgRNAs targeting exon 1 of Grxcr2 induced a 180-bp in-frame deletion, which was confirmed by sanger sequencing. (C) P3 cochlear explants were injectoporated to express Myc-GRXCR2 (Δ36–95). Two days later, tissues were fixed and stained with the Myc-antibody. Note Myc-GRXCR2 (Δ36–95) was concentrated at the base of the stereocilia. (D) Scanning electron microscope images showing auditory sensory epithelia of wild-type (upper panel), Grxcr2 –/– (middle panel), and Grxcr2 D 180/D180 (lower panel) mice at the age of P14. Note, stereocilia of Grxcr2 –/– mice were disorganized while the stereocilia were fairly normal in Grxcr2 D 180/D180 mice. Scale bars: left panel 5 μm, right panel 1 μm. (E,F) Cochlear whole mounts from wild-type and Grxcr2 D 180/D180 mice at the age of P7 (E) and 6 weeks (F) were stained for CLIC5. Stereocilia were visualized by staining with phalloidin (red). Note, CLIC5 staining did not show any significant change in Grxcr2 D 180/D180 hair cells. (G,H) Cochlear whole mounts from wild-type and Grxcr2 D 180/D180 mice at the age of P7 (G) and 6 weeks (H) were stained for taperin. Note, taperin was still concentrated at the base of stereocilia in Grxcr2 D 180/D180 hair cells. Scale bars: 5 μm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: N-Terminus of GRXCR2 Interacts With CLIC5 and Is Essential for Auditory Perception

    doi: 10.3389/fcell.2021.671364

    Figure Lengend Snippet: Disrupting the interaction between GRXCR2 and CLIC5 has minimal effects on stereocilia morphogenesis. (A) Amino acids from 36 to 95 in GRXCR2 are highly conserved in mammals. (B) Diagram of the strategy to generate Grxcr2 D 180/D180 mice. sgRNAs targeting exon 1 of Grxcr2 induced a 180-bp in-frame deletion, which was confirmed by sanger sequencing. (C) P3 cochlear explants were injectoporated to express Myc-GRXCR2 (Δ36–95). Two days later, tissues were fixed and stained with the Myc-antibody. Note Myc-GRXCR2 (Δ36–95) was concentrated at the base of the stereocilia. (D) Scanning electron microscope images showing auditory sensory epithelia of wild-type (upper panel), Grxcr2 –/– (middle panel), and Grxcr2 D 180/D180 (lower panel) mice at the age of P14. Note, stereocilia of Grxcr2 –/– mice were disorganized while the stereocilia were fairly normal in Grxcr2 D 180/D180 mice. Scale bars: left panel 5 μm, right panel 1 μm. (E,F) Cochlear whole mounts from wild-type and Grxcr2 D 180/D180 mice at the age of P7 (E) and 6 weeks (F) were stained for CLIC5. Stereocilia were visualized by staining with phalloidin (red). Note, CLIC5 staining did not show any significant change in Grxcr2 D 180/D180 hair cells. (G,H) Cochlear whole mounts from wild-type and Grxcr2 D 180/D180 mice at the age of P7 (G) and 6 weeks (H) were stained for taperin. Note, taperin was still concentrated at the base of stereocilia in Grxcr2 D 180/D180 hair cells. Scale bars: 5 μm.

    Article Snippet: Primary antibodies were as follows: anti-GRXCR2 (Cat# HPA059421, Sigma); anti-taperin (Cat# HPA020899, Sigma); anti-CLIC5 (Cat# ACL-025, Alomone labs).

    Techniques: Mouse Assay, Sequencing, Staining, Microscopy

    Disrupting the interaction between GRXCR2 and CLIC5 leads to hearing loss in mice. (A) Representative ABR traces to click stimuli in wild-type (black traces), Grxcr2 –/– (green traces), and Grxcr2 D 180/D180 mice (red traces). Note, Grxcr2 –/– mice had profound hearing loss while Grxcr2 D 180/D180 mice have moderate hearing loss. (B) ABR thresholds for click stimuli in 6-week-old wild-type, Grxcr2 –/– and Grxcr2 D 180/D180 mice. All values are represented as the mean ± SEM. *** p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: N-Terminus of GRXCR2 Interacts With CLIC5 and Is Essential for Auditory Perception

    doi: 10.3389/fcell.2021.671364

    Figure Lengend Snippet: Disrupting the interaction between GRXCR2 and CLIC5 leads to hearing loss in mice. (A) Representative ABR traces to click stimuli in wild-type (black traces), Grxcr2 –/– (green traces), and Grxcr2 D 180/D180 mice (red traces). Note, Grxcr2 –/– mice had profound hearing loss while Grxcr2 D 180/D180 mice have moderate hearing loss. (B) ABR thresholds for click stimuli in 6-week-old wild-type, Grxcr2 –/– and Grxcr2 D 180/D180 mice. All values are represented as the mean ± SEM. *** p

    Article Snippet: Primary antibodies were as follows: anti-GRXCR2 (Cat# HPA059421, Sigma); anti-taperin (Cat# HPA020899, Sigma); anti-CLIC5 (Cat# ACL-025, Alomone labs).

    Techniques: Mouse Assay

    Localization of CLIC1, CLIC4 and CLIC5 to the endoplasmic reticulum. (I) Isolated p3 neonatal cardiomyocytes were loaded with ER tracker (A, E, I), fixed, permeabilized, labeled with anti-CLIC1 (B), anti-CLIC4 (F), anti-CLIC5 (J) antibodies and further stained with DAPI (C, G, K). D, H and L are merge images showing colocalization of CLIC1 and CLIC4 to the endoplasmic reticulum whereas CLIC5 shows much less colocalization. D′, H′ and L′ are enlarged images of the squared region. (II) Bar graph representing percentage colocalization of CLIC1, CLIC4 and CLIC5 to endoplasmic reticulum ( n =3).

    Journal: Data in Brief

    Article Title: Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes

    doi: 10.1016/j.dib.2016.03.061

    Figure Lengend Snippet: Localization of CLIC1, CLIC4 and CLIC5 to the endoplasmic reticulum. (I) Isolated p3 neonatal cardiomyocytes were loaded with ER tracker (A, E, I), fixed, permeabilized, labeled with anti-CLIC1 (B), anti-CLIC4 (F), anti-CLIC5 (J) antibodies and further stained with DAPI (C, G, K). D, H and L are merge images showing colocalization of CLIC1 and CLIC4 to the endoplasmic reticulum whereas CLIC5 shows much less colocalization. D′, H′ and L′ are enlarged images of the squared region. (II) Bar graph representing percentage colocalization of CLIC1, CLIC4 and CLIC5 to endoplasmic reticulum ( n =3).

    Article Snippet: After loading the cells were fixed, permeabilized, and labeled with anti-CLIC1 [0.2 µg/mL, SC-271051 (lot no: E1711), Santa Cruz], CLIC4 [0.2 µg/mL, SC-135739 (lot no: D1911), Santa Cruz] and CLIC5 [0.2 µg/mL, ACL-025 (lot no: ANO102) Alomone lab] antibodies as mentioned above. (2) Adult rat cardiomyocytes: Dissociated cardiomyocytes were immediately transferred onto poly-l-lysine coated coverslips for 1 h at 4 °C and then loaded with 200 nM mitotracker for 10 min at 37 °C.

    Techniques: Isolation, Labeling, Staining

    Specificity of CLIC antibodies using CLIC1, CLIC4 and CLIC5 knock out (KO) mice heart lysates . (I) 50 µg of heart lysates from WT, clic1 −/− , clic4 −/− and clic5 −/− mouse were electrophoresed on 4–20% (w/v) SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-CLIC1, anti-CLIC4 and anti-CLIC5 antibodies. Absence of CLIC1, CLIC4 and CLIC5 specific bands in KO organ samples confirms the specificity of the antibody. Corresponding Ponceau S stained nitrocellulose membrane is shown as a loading control.

    Journal: Data in Brief

    Article Title: Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes

    doi: 10.1016/j.dib.2016.03.061

    Figure Lengend Snippet: Specificity of CLIC antibodies using CLIC1, CLIC4 and CLIC5 knock out (KO) mice heart lysates . (I) 50 µg of heart lysates from WT, clic1 −/− , clic4 −/− and clic5 −/− mouse were electrophoresed on 4–20% (w/v) SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-CLIC1, anti-CLIC4 and anti-CLIC5 antibodies. Absence of CLIC1, CLIC4 and CLIC5 specific bands in KO organ samples confirms the specificity of the antibody. Corresponding Ponceau S stained nitrocellulose membrane is shown as a loading control.

    Article Snippet: After loading the cells were fixed, permeabilized, and labeled with anti-CLIC1 [0.2 µg/mL, SC-271051 (lot no: E1711), Santa Cruz], CLIC4 [0.2 µg/mL, SC-135739 (lot no: D1911), Santa Cruz] and CLIC5 [0.2 µg/mL, ACL-025 (lot no: ANO102) Alomone lab] antibodies as mentioned above. (2) Adult rat cardiomyocytes: Dissociated cardiomyocytes were immediately transferred onto poly-l-lysine coated coverslips for 1 h at 4 °C and then loaded with 200 nM mitotracker for 10 min at 37 °C.

    Techniques: Knock-Out, Mouse Assay, SDS Page, Staining

    Chloride efflux is essential for NLRP3 inflammasome activation. a – c ELISA of IL-1β in LPS-primed BMDMs which were transferred to a basic NaCl saline (130 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) or chloride-free NaGluc saline (130 mM NaGluc, 5 mM KGluc, 1 mM MgGluc 2 , 1 mM CaGluc 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) and left stimulated with ATP a , MSU b , or nigericin c . d ELISA of IL-1β in LPS-primed BMDMs which were transferred to NaCl saline, NaBr saline (130 mM NaBr, 5 mM KBr, 1 mM MgBr 2 , 1 mM CaBr 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose), NaI saline (130 mM NaI, 5 mM KI, 1 mM MgI 2 , 1 mM CaI 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose), NaGluc saline or NaGlut (130 mM NaGlut, 5 mM KGlut, 1 mM MgGlut 2 , 1 mM CaGlut 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) saline for different time points. e , f Immunoblot analysis IL-1β and cleaved caspase-1 e or ELISA of IL-1β in supernatants f of LPS-primed Nlrp3 +/+ or Nlrp3 –/– BMDMs which transferred to NaCl, NaBr or NaI saline for 3 h. g ELISA of IL-1β in supernatants of LPS-primed Clic4 –/– BMDMs transfected siRNA against Clic1 and Clic5 and left stimulated with nigericin for 30 min or transferred to NaCl, NaBr or NaI saline for 3 h. Data are from three independent experiments with biological duplicates in each ( a – d , f , g ; mean ± SEM of n = 6) or are representative of three independent experiments e . Student’s t -test a – c , f , g , *** P

    Journal: Nature Communications

    Article Title: CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation

    doi: 10.1038/s41467-017-00227-x

    Figure Lengend Snippet: Chloride efflux is essential for NLRP3 inflammasome activation. a – c ELISA of IL-1β in LPS-primed BMDMs which were transferred to a basic NaCl saline (130 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) or chloride-free NaGluc saline (130 mM NaGluc, 5 mM KGluc, 1 mM MgGluc 2 , 1 mM CaGluc 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) and left stimulated with ATP a , MSU b , or nigericin c . d ELISA of IL-1β in LPS-primed BMDMs which were transferred to NaCl saline, NaBr saline (130 mM NaBr, 5 mM KBr, 1 mM MgBr 2 , 1 mM CaBr 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose), NaI saline (130 mM NaI, 5 mM KI, 1 mM MgI 2 , 1 mM CaI 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose), NaGluc saline or NaGlut (130 mM NaGlut, 5 mM KGlut, 1 mM MgGlut 2 , 1 mM CaGlut 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) saline for different time points. e , f Immunoblot analysis IL-1β and cleaved caspase-1 e or ELISA of IL-1β in supernatants f of LPS-primed Nlrp3 +/+ or Nlrp3 –/– BMDMs which transferred to NaCl, NaBr or NaI saline for 3 h. g ELISA of IL-1β in supernatants of LPS-primed Clic4 –/– BMDMs transfected siRNA against Clic1 and Clic5 and left stimulated with nigericin for 30 min or transferred to NaCl, NaBr or NaI saline for 3 h. Data are from three independent experiments with biological duplicates in each ( a – d , f , g ; mean ± SEM of n = 6) or are representative of three independent experiments e . Student’s t -test a – c , f , g , *** P

    Article Snippet: Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection

    Clics are important for NLRP3 inflammasome assembly. a Immunoblot analysis of ASC oligomerization in lysates of LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 and left stimulated with ATP or nigericin. b Immunoprecipitation (IP) and immunoblot analysis of the interaction of endogenous NLRP3 and ASC in LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 , and left stimulated with ATP or nigericin. c IP and immunoblot analysis of the interaction of endogenous NLRP3 and NEK7 in LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 , and left stimulated with ATP or nigericin. d Model for the function and mechanism of Clics and chloride efflux in NLRP3 inflammasome activation. Data are representative of three independent experiments a – c

    Journal: Nature Communications

    Article Title: CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation

    doi: 10.1038/s41467-017-00227-x

    Figure Lengend Snippet: Clics are important for NLRP3 inflammasome assembly. a Immunoblot analysis of ASC oligomerization in lysates of LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 and left stimulated with ATP or nigericin. b Immunoprecipitation (IP) and immunoblot analysis of the interaction of endogenous NLRP3 and ASC in LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 , and left stimulated with ATP or nigericin. c IP and immunoblot analysis of the interaction of endogenous NLRP3 and NEK7 in LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 , and left stimulated with ATP or nigericin. d Model for the function and mechanism of Clics and chloride efflux in NLRP3 inflammasome activation. Data are representative of three independent experiments a – c

    Article Snippet: Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs.

    Techniques: Transfection, Immunoprecipitation, Activation Assay

    Clics act downstream of potassium efflux or mitochondrial damage to promote NLRP3 activation. a , b Qualification of the decrease of intracellular potassium in LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 a or Nlrp3 –/– BMDMs tranfected with siRNA against Clic1 , Clic4 and Clic5 b and left stimulated with ATP or nigericin. c ELISA of IL-1β in supernatants of LPS-primed Clic4 –/– BMDMs transfected siRNA against Clic1 and Clic5 and left transferred to potassium-free saline (135 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) for 3 h. d Confocal microscopy analysis of LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 and then left stimulated with nigericin, followed by staining with Mitotracker red and DAPI. e ELISA of IL-1β in supernatants of LPS-primed BMDMs transfected stimulated with nigericin or transferred to NaBr, NaI saline or potassium-free saline with or without the presence of indicated doses of MnTBAP. Data are from three independent experiments with biological duplicates in each ( a – c , e ; mean ± SEM of n = 6) or are representative of three independent experiments d . Two-way ANOVA e , Student’s t -test a – c , *** P

    Journal: Nature Communications

    Article Title: CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation

    doi: 10.1038/s41467-017-00227-x

    Figure Lengend Snippet: Clics act downstream of potassium efflux or mitochondrial damage to promote NLRP3 activation. a , b Qualification of the decrease of intracellular potassium in LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 a or Nlrp3 –/– BMDMs tranfected with siRNA against Clic1 , Clic4 and Clic5 b and left stimulated with ATP or nigericin. c ELISA of IL-1β in supernatants of LPS-primed Clic4 –/– BMDMs transfected siRNA against Clic1 and Clic5 and left transferred to potassium-free saline (135 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 20 mM HEPES (pH 7.4), 1 mg/ml BSA, 10 mM glucose) for 3 h. d Confocal microscopy analysis of LPS-primed Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 and then left stimulated with nigericin, followed by staining with Mitotracker red and DAPI. e ELISA of IL-1β in supernatants of LPS-primed BMDMs transfected stimulated with nigericin or transferred to NaBr, NaI saline or potassium-free saline with or without the presence of indicated doses of MnTBAP. Data are from three independent experiments with biological duplicates in each ( a – c , e ; mean ± SEM of n = 6) or are representative of three independent experiments d . Two-way ANOVA e , Student’s t -test a – c , *** P

    Article Snippet: Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs.

    Techniques: Activated Clotting Time Assay, Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining

    Inhibition of Clics suppresses NLRP3 inflammasome activation. a ELISA of IL-1β in culture supernatants of LPS-primed BMDMs transfected with siRNA against Clic1, 4, or 5 and left stimulated with nigericin. b ELISA of IL-1β in culture supernatants of LPS-primed Clic1 –/– , Clic4 –/– , or Clic5 –/– BMDMs stimulated with nigericin. c ELISA analysis of IL-1β in culture supernatants of LPS-primed wildtype or Clic4 –/– BMDMs transfected with siRNA against Clic1, Clic5 , or both of them and left stimulated with nigericin. d , e ELISA d or Immunoblot e analysis of IL-1β in culture supernatants of LPS-primed wildtype or Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 , and left stimulated with nigericin, MSU, or ATP. f ELISA of IL-1β in culture supernatants of LPS-primed wildtype or Clic1 –/– BMDMs transfected with siRNA against Clic4 and Clic5 , and left stimulated with nigericin, MSU, or ATP. g ELISA of IL-1β in culture supernatants of LPS-primed wild type or Clic5 –/– BMDMs transfected with siRNA against Clic1 and Clic4 and left stimulated with nigericin, MSU, or ATP. Data are from three independent experiments with biological duplicates in each ( a – d , f , g ; mean ± SEM of n = 6) or are representative of three independent experiments e . Student’s t -test a - d , f , g , ** P

    Journal: Nature Communications

    Article Title: CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation

    doi: 10.1038/s41467-017-00227-x

    Figure Lengend Snippet: Inhibition of Clics suppresses NLRP3 inflammasome activation. a ELISA of IL-1β in culture supernatants of LPS-primed BMDMs transfected with siRNA against Clic1, 4, or 5 and left stimulated with nigericin. b ELISA of IL-1β in culture supernatants of LPS-primed Clic1 –/– , Clic4 –/– , or Clic5 –/– BMDMs stimulated with nigericin. c ELISA analysis of IL-1β in culture supernatants of LPS-primed wildtype or Clic4 –/– BMDMs transfected with siRNA against Clic1, Clic5 , or both of them and left stimulated with nigericin. d , e ELISA d or Immunoblot e analysis of IL-1β in culture supernatants of LPS-primed wildtype or Clic4 –/– BMDMs transfected with siRNA against Clic1 and Clic5 , and left stimulated with nigericin, MSU, or ATP. f ELISA of IL-1β in culture supernatants of LPS-primed wildtype or Clic1 –/– BMDMs transfected with siRNA against Clic4 and Clic5 , and left stimulated with nigericin, MSU, or ATP. g ELISA of IL-1β in culture supernatants of LPS-primed wild type or Clic5 –/– BMDMs transfected with siRNA against Clic1 and Clic4 and left stimulated with nigericin, MSU, or ATP. Data are from three independent experiments with biological duplicates in each ( a – d , f , g ; mean ± SEM of n = 6) or are representative of three independent experiments e . Student’s t -test a - d , f , g , ** P

    Article Snippet: Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs.

    Techniques: Inhibition, Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection

    Clics-dependent chloride efflux during NLRP3 inflammasome activation. a – c Qualification of the decrease of intracellular chloride in wild type or Nlrp3 –/– BMDMs at different time points after nigericin a , ATP b , or poly A:T c treatment. d , e Qualification of the decrease of intracellular chloride in BMDMs at different time points after nigericin d or ATP e treatment with or without the pretreatment with IAA94 (150 μM). f , g Qualification of the decrease of intracellular chloride in Clic4 –/– BMDMs transfected siRNA against Clic1 and Clic5 at different time points after nigericin f or ATP g treatment. Data are from three independent experiments with biological duplicates in each (mean ± SEM of n = 6). Student’s t -test, * P

    Journal: Nature Communications

    Article Title: CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation

    doi: 10.1038/s41467-017-00227-x

    Figure Lengend Snippet: Clics-dependent chloride efflux during NLRP3 inflammasome activation. a – c Qualification of the decrease of intracellular chloride in wild type or Nlrp3 –/– BMDMs at different time points after nigericin a , ATP b , or poly A:T c treatment. d , e Qualification of the decrease of intracellular chloride in BMDMs at different time points after nigericin d or ATP e treatment with or without the pretreatment with IAA94 (150 μM). f , g Qualification of the decrease of intracellular chloride in Clic4 –/– BMDMs transfected siRNA against Clic1 and Clic5 at different time points after nigericin f or ATP g treatment. Data are from three independent experiments with biological duplicates in each (mean ± SEM of n = 6). Student’s t -test, * P

    Article Snippet: Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs.

    Techniques: Activation Assay, Transfection

    Clics are important for NLRP3 inflammasome activation in vivo. a , b FACS analysis of neutrophil numbers a or ELISA b of IL-1β in the peritoneal cavity of C57BL/6J mice intraperitoneally injected with MSU (1 mg per mouse) with or without IAA94 (50 mg/kg of body weight). n = 6 or 7 per group. c qPCR analysis of Clic5 expression in peritoneal macrophages from Clic4 –/– mice intraperitoneally injected with nanoparticle-encapsulated siRNA (40 μg per mouse). d , e FACS analysis of neutrophil numbers d or ELISA e of IL-1β in the peritoneal cavity of Clic4 –/– mice intraperitoneally injected with nanoparticle-encapsulated siRNA (40 μg per mouse) and MSU (1 mg per mouse). n = 5–7 per group. Data are shown as mean ± SEM and are representative of two independent experiments. Student’s t -test, ** P

    Journal: Nature Communications

    Article Title: CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation

    doi: 10.1038/s41467-017-00227-x

    Figure Lengend Snippet: Clics are important for NLRP3 inflammasome activation in vivo. a , b FACS analysis of neutrophil numbers a or ELISA b of IL-1β in the peritoneal cavity of C57BL/6J mice intraperitoneally injected with MSU (1 mg per mouse) with or without IAA94 (50 mg/kg of body weight). n = 6 or 7 per group. c qPCR analysis of Clic5 expression in peritoneal macrophages from Clic4 –/– mice intraperitoneally injected with nanoparticle-encapsulated siRNA (40 μg per mouse). d , e FACS analysis of neutrophil numbers d or ELISA e of IL-1β in the peritoneal cavity of Clic4 –/– mice intraperitoneally injected with nanoparticle-encapsulated siRNA (40 μg per mouse) and MSU (1 mg per mouse). n = 5–7 per group. Data are shown as mean ± SEM and are representative of two independent experiments. Student’s t -test, ** P

    Article Snippet: Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs.

    Techniques: Activation Assay, In Vivo, FACS, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Expressing