rabbit anti clc 1  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti clc 1
    Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing <t>Flag-CLC-1</t> in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p
    Rabbit Anti Clc 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti clc 1/product/Alomone Labs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti clc 1 - by Bioz Stars, 2022-08
    90/100 stars

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    1) Product Images from "FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane"

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123783

    Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p
    Figure Legend Snippet: Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Molecular Weight, Labeling

    Sucrose gradient fractionation analyses of FKBP8 localization. Cell homogenates from HEK293T cells transfected with the indicated cDNA constructs were analyzed by sucrose density gradient centrifugation. Total homogenates ( Total ) were ultracentrifuged and separated into the supernatant ( Cytosol ) and the membrane pellet ( Memb ) fractions. The membrane pellet fraction was further sedimented through a discontinuous sucrose gradient and subsequently divided into 8 fractions, with the density gradient increasing from fraction 1 toward fraction 8. Specific markers for distinct subcellular compartments were chosen by studying 3 endogenous proteins in HEK293T cells: cadherin (plasma membrane protein), calnexin (ER-resident membrane-associated protein), and GAPDH (cytosolic protein). ( A ) ( Left panels ) Representative immunoblots showing the subcellular fractionation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Densitometric quantification of the relative distribution (with respect to the total signal) in different fractions for each membrane-associated protein analyzed on the corresponding gel to the left; ( B ) Statistical analyses of the ratio of protein signal intensities at fractions 3 and 4 to those at fraction 8 [F(3+4)/F8] for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( C ) Representative sucrose gradient fractionation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p
    Figure Legend Snippet: Sucrose gradient fractionation analyses of FKBP8 localization. Cell homogenates from HEK293T cells transfected with the indicated cDNA constructs were analyzed by sucrose density gradient centrifugation. Total homogenates ( Total ) were ultracentrifuged and separated into the supernatant ( Cytosol ) and the membrane pellet ( Memb ) fractions. The membrane pellet fraction was further sedimented through a discontinuous sucrose gradient and subsequently divided into 8 fractions, with the density gradient increasing from fraction 1 toward fraction 8. Specific markers for distinct subcellular compartments were chosen by studying 3 endogenous proteins in HEK293T cells: cadherin (plasma membrane protein), calnexin (ER-resident membrane-associated protein), and GAPDH (cytosolic protein). ( A ) ( Left panels ) Representative immunoblots showing the subcellular fractionation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Densitometric quantification of the relative distribution (with respect to the total signal) in different fractions for each membrane-associated protein analyzed on the corresponding gel to the left; ( B ) Statistical analyses of the ratio of protein signal intensities at fractions 3 and 4 to those at fraction 8 [F(3+4)/F8] for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( C ) Representative sucrose gradient fractionation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p

    Techniques Used: Fractionation, Transfection, Construct, Gradient Centrifugation, Western Blot, Expressing

    Localization of FKBP8 at the Golgi apparatus. ( A ) Differential centrifugation analysis of lysates from HEK293T cells over-expressing GFP-tagged trans -Golgi network 38 (GFP-TGN38). ( Left ) Representative immunoblots showing the fractionation pattern of GFP-TGN38 and the indicated endogenous proteins. Total homogenates ( Total ) were separated by sequential 1000× g (1 k) and 10,000× g (10 k), and 100,000× g (100 k) centrifugation. The pellets retrieved from each of the 3 centrifugation forces, as well as the supernatant ( Cytosol ) of the 100 k ultracentrifugation, were then subject to electrophoresis analyses. The endogenous cis -Golgi network protein GM130 serves as the specific marker for the Golgi apparatus of HEK293T cells. ( Right ) Densitometric quantification of the ratio of the protein signal intensity at the 100 k fraction to that at the 1 k fraction (100 k/1 k) ( n = 3). No statistical comparisons were preformed; ( B ) ( Left panels ) Representative immunoblots showing the differential centrifugation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Quantification of the relative distribution (with respect to the total signal) in the three pellet fractions for membrane-associated proteins analyzed on the gel to the left; ( C ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( D ) Representative differential centrifugation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( E ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 3). The asterisk denotes a significant difference from the control (*, t -test: p
    Figure Legend Snippet: Localization of FKBP8 at the Golgi apparatus. ( A ) Differential centrifugation analysis of lysates from HEK293T cells over-expressing GFP-tagged trans -Golgi network 38 (GFP-TGN38). ( Left ) Representative immunoblots showing the fractionation pattern of GFP-TGN38 and the indicated endogenous proteins. Total homogenates ( Total ) were separated by sequential 1000× g (1 k) and 10,000× g (10 k), and 100,000× g (100 k) centrifugation. The pellets retrieved from each of the 3 centrifugation forces, as well as the supernatant ( Cytosol ) of the 100 k ultracentrifugation, were then subject to electrophoresis analyses. The endogenous cis -Golgi network protein GM130 serves as the specific marker for the Golgi apparatus of HEK293T cells. ( Right ) Densitometric quantification of the ratio of the protein signal intensity at the 100 k fraction to that at the 1 k fraction (100 k/1 k) ( n = 3). No statistical comparisons were preformed; ( B ) ( Left panels ) Representative immunoblots showing the differential centrifugation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Quantification of the relative distribution (with respect to the total signal) in the three pellet fractions for membrane-associated proteins analyzed on the gel to the left; ( C ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( D ) Representative differential centrifugation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( E ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 3). The asterisk denotes a significant difference from the control (*, t -test: p

    Techniques Used: Centrifugation, Expressing, Western Blot, Fractionation, Electrophoresis, Marker

    Subcellular localization of endogenous FKBP8 in HEK293T cells. ( A ) Surface biotinylation analysis of endogenous FKBP8 in the absence or presence of Flag-CLC-1 over-expression ( n = 3); ( B ) Representative biotinylation result of endogenous calnexin in the absence or presence of Flag-CLC-1 over-expression; ( C ) Representative sucrose density gradient centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence ( Flag-vector ) or presence ( Flag-CLC-1 ) of Flag-CLC-1 over-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4); ( E ) Representative differential centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence or presence of Flag-CLC-1 over-expression; ( F ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p
    Figure Legend Snippet: Subcellular localization of endogenous FKBP8 in HEK293T cells. ( A ) Surface biotinylation analysis of endogenous FKBP8 in the absence or presence of Flag-CLC-1 over-expression ( n = 3); ( B ) Representative biotinylation result of endogenous calnexin in the absence or presence of Flag-CLC-1 over-expression; ( C ) Representative sucrose density gradient centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence ( Flag-vector ) or presence ( Flag-CLC-1 ) of Flag-CLC-1 over-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4); ( E ) Representative differential centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence or presence of Flag-CLC-1 over-expression; ( F ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p

    Techniques Used: Over Expression, Gradient Centrifugation, Plasmid Preparation, Centrifugation

    Plasma membrane-localization of endogenous FKBP8 in skeletal muscle. ( A ) Representative protein signals of endogenous FKBP8 in human (HEK293T) cells ( open triangle ) and rat skeletal muscle ( filled triangle ); ( B ) Representative sucrose fractionation pattern of endogenous FKBP8, CLC-1, cadherin, calnexin, and GAPDH in rat skeletal muscle; ( C ) Representative immunoblot showing the presence of rat muscle CLC-1, cadherin, and FBKP8 at the plasma membrane. Plasma membrane fraction ( PM ) was isolated with an aqueous two-phase system. Note that GAPDH and calnexin are found in the total protein fraction ( Total ), but not in the plasma membrane fraction; ( D , E ) Co-immunoprecipitation of FKBP8 and CLC-1 in skeletal muscle. Muscle lysates were subject to immunoprecipitation ( IP ) with the anti-CLC-1 ( D ) or anti-FKBP8 ( E ) antibody, followed by immunoblotting with the indicated antibodies. Rabbit IgG was employed to verify the specificity of immunoprecipitation. Corresponding expression levels of CLC-1 and FKBP8 in the lysates are shown in the Input lane. Input represents about 10% of the total protein used for immunoprecipitation. h.c. : IgG heavy chain.
    Figure Legend Snippet: Plasma membrane-localization of endogenous FKBP8 in skeletal muscle. ( A ) Representative protein signals of endogenous FKBP8 in human (HEK293T) cells ( open triangle ) and rat skeletal muscle ( filled triangle ); ( B ) Representative sucrose fractionation pattern of endogenous FKBP8, CLC-1, cadherin, calnexin, and GAPDH in rat skeletal muscle; ( C ) Representative immunoblot showing the presence of rat muscle CLC-1, cadherin, and FBKP8 at the plasma membrane. Plasma membrane fraction ( PM ) was isolated with an aqueous two-phase system. Note that GAPDH and calnexin are found in the total protein fraction ( Total ), but not in the plasma membrane fraction; ( D , E ) Co-immunoprecipitation of FKBP8 and CLC-1 in skeletal muscle. Muscle lysates were subject to immunoprecipitation ( IP ) with the anti-CLC-1 ( D ) or anti-FKBP8 ( E ) antibody, followed by immunoblotting with the indicated antibodies. Rabbit IgG was employed to verify the specificity of immunoprecipitation. Corresponding expression levels of CLC-1 and FKBP8 in the lysates are shown in the Input lane. Input represents about 10% of the total protein used for immunoprecipitation. h.c. : IgG heavy chain.

    Techniques Used: Fractionation, Isolation, Immunoprecipitation, Expressing

    FKBP8 promotes surface protein stability. ( A , B ) Representative immunoblots showing the time course of surface Flag-CLC-1 protein turn-over in the absence ( A ) or presence ( B ) of Myc-FKBP8 over-expression. Transfected HEK293T cells were subject to the indicated durations of treatment with the Golgi trafficking inhibitor brefeldin A (BFA) (5 μg/mL), followed by surface biotinylation analyses; ( C – E ) Quantification of surface Flag-CLC-1 protein stability in the absence ( black ) or presence ( red ) of Myc-FKBP8 over-expression ( n = 6–8); ( C ) Linear plot of the turn-over kinetics of surface CLC-1 protein densities. Protein density was standardized as the ratio of surface Flag-CLC-1 signal to the cognate total GAPDH signal, followed by normalization to the corresponding control at 0 hr; ( D ) Semi-logarithmic plot of the same data points, followed by linear-regression analyses ( solid lines ); ( E ) Statistical analyses of surface CLC-1 protein half-life. The surface protein turn-over time course determined from each experiment was individually plotted on a semi-logarithmic scale for linear-regression analysis, and independent protein half-life values were pooled together for statistical comparisons. The asterisk denotes a significant difference from the control (*, t -test: p
    Figure Legend Snippet: FKBP8 promotes surface protein stability. ( A , B ) Representative immunoblots showing the time course of surface Flag-CLC-1 protein turn-over in the absence ( A ) or presence ( B ) of Myc-FKBP8 over-expression. Transfected HEK293T cells were subject to the indicated durations of treatment with the Golgi trafficking inhibitor brefeldin A (BFA) (5 μg/mL), followed by surface biotinylation analyses; ( C – E ) Quantification of surface Flag-CLC-1 protein stability in the absence ( black ) or presence ( red ) of Myc-FKBP8 over-expression ( n = 6–8); ( C ) Linear plot of the turn-over kinetics of surface CLC-1 protein densities. Protein density was standardized as the ratio of surface Flag-CLC-1 signal to the cognate total GAPDH signal, followed by normalization to the corresponding control at 0 hr; ( D ) Semi-logarithmic plot of the same data points, followed by linear-regression analyses ( solid lines ); ( E ) Statistical analyses of surface CLC-1 protein half-life. The surface protein turn-over time course determined from each experiment was individually plotted on a semi-logarithmic scale for linear-regression analysis, and independent protein half-life values were pooled together for statistical comparisons. The asterisk denotes a significant difference from the control (*, t -test: p

    Techniques Used: Western Blot, Over Expression, Transfection

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    Alomone Labs rabbit anti clc 1
    Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing <t>Flag-CLC-1</t> in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p
    Rabbit Anti Clc 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti clc 1/product/Alomone Labs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti clc 1 - by Bioz Stars, 2022-08
    90/100 stars
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    Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p

    Journal: International Journal of Molecular Sciences

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    doi: 10.3390/ijms19123783

    Figure Lengend Snippet: Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p

    Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using rabbit anti-Aha1 (1:2500; Thermo Scientific, Waltham, MA, USA), mouse anti-cadherin (1:1000; Abcam, Cambridge, MA, USA), mouse anti-calnexin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CLC-1 (1:1000; Alomone, Jerusalem, Israel), rabbit anti-Flag (1:5000; Sigma-Aldrich), rabbit anti-FKBP8 (1:5000; GeneTex, Hsinchu, Taiwan), rabbit anti-GAPDH (1:8000; GeneTex), rat anti-HA (1:5000; Roche Applied Science), rabbit anti-GFP (1:5000; Abcam), mouse anti-GM130 (1:1000; BD Biosciences, San Jose, CA, USA), rabbit anti-HOP (1:10,000; Abcam), rabbit anti-Hsc70 (1:750; Abcam), rabbit anti- Hsp90β (1:500; Abcam), mouse anti-Myc (clone 9E10), or mouse anti-Na+ -K+-ATPase (1:1000; Thermo Scientific) antibodies.

    Techniques: Expressing, Western Blot, Plasmid Preparation, Molecular Weight, Labeling

    Sucrose gradient fractionation analyses of FKBP8 localization. Cell homogenates from HEK293T cells transfected with the indicated cDNA constructs were analyzed by sucrose density gradient centrifugation. Total homogenates ( Total ) were ultracentrifuged and separated into the supernatant ( Cytosol ) and the membrane pellet ( Memb ) fractions. The membrane pellet fraction was further sedimented through a discontinuous sucrose gradient and subsequently divided into 8 fractions, with the density gradient increasing from fraction 1 toward fraction 8. Specific markers for distinct subcellular compartments were chosen by studying 3 endogenous proteins in HEK293T cells: cadherin (plasma membrane protein), calnexin (ER-resident membrane-associated protein), and GAPDH (cytosolic protein). ( A ) ( Left panels ) Representative immunoblots showing the subcellular fractionation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Densitometric quantification of the relative distribution (with respect to the total signal) in different fractions for each membrane-associated protein analyzed on the corresponding gel to the left; ( B ) Statistical analyses of the ratio of protein signal intensities at fractions 3 and 4 to those at fraction 8 [F(3+4)/F8] for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( C ) Representative sucrose gradient fractionation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p

    Journal: International Journal of Molecular Sciences

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    doi: 10.3390/ijms19123783

    Figure Lengend Snippet: Sucrose gradient fractionation analyses of FKBP8 localization. Cell homogenates from HEK293T cells transfected with the indicated cDNA constructs were analyzed by sucrose density gradient centrifugation. Total homogenates ( Total ) were ultracentrifuged and separated into the supernatant ( Cytosol ) and the membrane pellet ( Memb ) fractions. The membrane pellet fraction was further sedimented through a discontinuous sucrose gradient and subsequently divided into 8 fractions, with the density gradient increasing from fraction 1 toward fraction 8. Specific markers for distinct subcellular compartments were chosen by studying 3 endogenous proteins in HEK293T cells: cadherin (plasma membrane protein), calnexin (ER-resident membrane-associated protein), and GAPDH (cytosolic protein). ( A ) ( Left panels ) Representative immunoblots showing the subcellular fractionation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Densitometric quantification of the relative distribution (with respect to the total signal) in different fractions for each membrane-associated protein analyzed on the corresponding gel to the left; ( B ) Statistical analyses of the ratio of protein signal intensities at fractions 3 and 4 to those at fraction 8 [F(3+4)/F8] for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( C ) Representative sucrose gradient fractionation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p

    Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using rabbit anti-Aha1 (1:2500; Thermo Scientific, Waltham, MA, USA), mouse anti-cadherin (1:1000; Abcam, Cambridge, MA, USA), mouse anti-calnexin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CLC-1 (1:1000; Alomone, Jerusalem, Israel), rabbit anti-Flag (1:5000; Sigma-Aldrich), rabbit anti-FKBP8 (1:5000; GeneTex, Hsinchu, Taiwan), rabbit anti-GAPDH (1:8000; GeneTex), rat anti-HA (1:5000; Roche Applied Science), rabbit anti-GFP (1:5000; Abcam), mouse anti-GM130 (1:1000; BD Biosciences, San Jose, CA, USA), rabbit anti-HOP (1:10,000; Abcam), rabbit anti-Hsc70 (1:750; Abcam), rabbit anti- Hsp90β (1:500; Abcam), mouse anti-Myc (clone 9E10), or mouse anti-Na+ -K+-ATPase (1:1000; Thermo Scientific) antibodies.

    Techniques: Fractionation, Transfection, Construct, Gradient Centrifugation, Western Blot, Expressing

    Localization of FKBP8 at the Golgi apparatus. ( A ) Differential centrifugation analysis of lysates from HEK293T cells over-expressing GFP-tagged trans -Golgi network 38 (GFP-TGN38). ( Left ) Representative immunoblots showing the fractionation pattern of GFP-TGN38 and the indicated endogenous proteins. Total homogenates ( Total ) were separated by sequential 1000× g (1 k) and 10,000× g (10 k), and 100,000× g (100 k) centrifugation. The pellets retrieved from each of the 3 centrifugation forces, as well as the supernatant ( Cytosol ) of the 100 k ultracentrifugation, were then subject to electrophoresis analyses. The endogenous cis -Golgi network protein GM130 serves as the specific marker for the Golgi apparatus of HEK293T cells. ( Right ) Densitometric quantification of the ratio of the protein signal intensity at the 100 k fraction to that at the 1 k fraction (100 k/1 k) ( n = 3). No statistical comparisons were preformed; ( B ) ( Left panels ) Representative immunoblots showing the differential centrifugation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Quantification of the relative distribution (with respect to the total signal) in the three pellet fractions for membrane-associated proteins analyzed on the gel to the left; ( C ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( D ) Representative differential centrifugation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( E ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 3). The asterisk denotes a significant difference from the control (*, t -test: p

    Journal: International Journal of Molecular Sciences

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    doi: 10.3390/ijms19123783

    Figure Lengend Snippet: Localization of FKBP8 at the Golgi apparatus. ( A ) Differential centrifugation analysis of lysates from HEK293T cells over-expressing GFP-tagged trans -Golgi network 38 (GFP-TGN38). ( Left ) Representative immunoblots showing the fractionation pattern of GFP-TGN38 and the indicated endogenous proteins. Total homogenates ( Total ) were separated by sequential 1000× g (1 k) and 10,000× g (10 k), and 100,000× g (100 k) centrifugation. The pellets retrieved from each of the 3 centrifugation forces, as well as the supernatant ( Cytosol ) of the 100 k ultracentrifugation, were then subject to electrophoresis analyses. The endogenous cis -Golgi network protein GM130 serves as the specific marker for the Golgi apparatus of HEK293T cells. ( Right ) Densitometric quantification of the ratio of the protein signal intensity at the 100 k fraction to that at the 1 k fraction (100 k/1 k) ( n = 3). No statistical comparisons were preformed; ( B ) ( Left panels ) Representative immunoblots showing the differential centrifugation pattern of Flag-CLC-1 in the absence or presence of Myc-FKBP8 co-expression. ( Right panels ) Quantification of the relative distribution (with respect to the total signal) in the three pellet fractions for membrane-associated proteins analyzed on the gel to the left; ( C ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Myc-FKBP8 ( n = 3); ( D ) Representative differential centrifugation pattern of Myc-FKBP8 in the absence or presence of Flag-CLC-1 co-expression; ( E ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 3). The asterisk denotes a significant difference from the control (*, t -test: p

    Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using rabbit anti-Aha1 (1:2500; Thermo Scientific, Waltham, MA, USA), mouse anti-cadherin (1:1000; Abcam, Cambridge, MA, USA), mouse anti-calnexin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CLC-1 (1:1000; Alomone, Jerusalem, Israel), rabbit anti-Flag (1:5000; Sigma-Aldrich), rabbit anti-FKBP8 (1:5000; GeneTex, Hsinchu, Taiwan), rabbit anti-GAPDH (1:8000; GeneTex), rat anti-HA (1:5000; Roche Applied Science), rabbit anti-GFP (1:5000; Abcam), mouse anti-GM130 (1:1000; BD Biosciences, San Jose, CA, USA), rabbit anti-HOP (1:10,000; Abcam), rabbit anti-Hsc70 (1:750; Abcam), rabbit anti- Hsp90β (1:500; Abcam), mouse anti-Myc (clone 9E10), or mouse anti-Na+ -K+-ATPase (1:1000; Thermo Scientific) antibodies.

    Techniques: Centrifugation, Expressing, Western Blot, Fractionation, Electrophoresis, Marker

    Subcellular localization of endogenous FKBP8 in HEK293T cells. ( A ) Surface biotinylation analysis of endogenous FKBP8 in the absence or presence of Flag-CLC-1 over-expression ( n = 3); ( B ) Representative biotinylation result of endogenous calnexin in the absence or presence of Flag-CLC-1 over-expression; ( C ) Representative sucrose density gradient centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence ( Flag-vector ) or presence ( Flag-CLC-1 ) of Flag-CLC-1 over-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4); ( E ) Representative differential centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence or presence of Flag-CLC-1 over-expression; ( F ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p

    Journal: International Journal of Molecular Sciences

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    doi: 10.3390/ijms19123783

    Figure Lengend Snippet: Subcellular localization of endogenous FKBP8 in HEK293T cells. ( A ) Surface biotinylation analysis of endogenous FKBP8 in the absence or presence of Flag-CLC-1 over-expression ( n = 3); ( B ) Representative biotinylation result of endogenous calnexin in the absence or presence of Flag-CLC-1 over-expression; ( C ) Representative sucrose density gradient centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence ( Flag-vector ) or presence ( Flag-CLC-1 ) of Flag-CLC-1 over-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4); ( E ) Representative differential centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence or presence of Flag-CLC-1 over-expression; ( F ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p

    Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using rabbit anti-Aha1 (1:2500; Thermo Scientific, Waltham, MA, USA), mouse anti-cadherin (1:1000; Abcam, Cambridge, MA, USA), mouse anti-calnexin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CLC-1 (1:1000; Alomone, Jerusalem, Israel), rabbit anti-Flag (1:5000; Sigma-Aldrich), rabbit anti-FKBP8 (1:5000; GeneTex, Hsinchu, Taiwan), rabbit anti-GAPDH (1:8000; GeneTex), rat anti-HA (1:5000; Roche Applied Science), rabbit anti-GFP (1:5000; Abcam), mouse anti-GM130 (1:1000; BD Biosciences, San Jose, CA, USA), rabbit anti-HOP (1:10,000; Abcam), rabbit anti-Hsc70 (1:750; Abcam), rabbit anti- Hsp90β (1:500; Abcam), mouse anti-Myc (clone 9E10), or mouse anti-Na+ -K+-ATPase (1:1000; Thermo Scientific) antibodies.

    Techniques: Over Expression, Gradient Centrifugation, Plasmid Preparation, Centrifugation

    Plasma membrane-localization of endogenous FKBP8 in skeletal muscle. ( A ) Representative protein signals of endogenous FKBP8 in human (HEK293T) cells ( open triangle ) and rat skeletal muscle ( filled triangle ); ( B ) Representative sucrose fractionation pattern of endogenous FKBP8, CLC-1, cadherin, calnexin, and GAPDH in rat skeletal muscle; ( C ) Representative immunoblot showing the presence of rat muscle CLC-1, cadherin, and FBKP8 at the plasma membrane. Plasma membrane fraction ( PM ) was isolated with an aqueous two-phase system. Note that GAPDH and calnexin are found in the total protein fraction ( Total ), but not in the plasma membrane fraction; ( D , E ) Co-immunoprecipitation of FKBP8 and CLC-1 in skeletal muscle. Muscle lysates were subject to immunoprecipitation ( IP ) with the anti-CLC-1 ( D ) or anti-FKBP8 ( E ) antibody, followed by immunoblotting with the indicated antibodies. Rabbit IgG was employed to verify the specificity of immunoprecipitation. Corresponding expression levels of CLC-1 and FKBP8 in the lysates are shown in the Input lane. Input represents about 10% of the total protein used for immunoprecipitation. h.c. : IgG heavy chain.

    Journal: International Journal of Molecular Sciences

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    doi: 10.3390/ijms19123783

    Figure Lengend Snippet: Plasma membrane-localization of endogenous FKBP8 in skeletal muscle. ( A ) Representative protein signals of endogenous FKBP8 in human (HEK293T) cells ( open triangle ) and rat skeletal muscle ( filled triangle ); ( B ) Representative sucrose fractionation pattern of endogenous FKBP8, CLC-1, cadherin, calnexin, and GAPDH in rat skeletal muscle; ( C ) Representative immunoblot showing the presence of rat muscle CLC-1, cadherin, and FBKP8 at the plasma membrane. Plasma membrane fraction ( PM ) was isolated with an aqueous two-phase system. Note that GAPDH and calnexin are found in the total protein fraction ( Total ), but not in the plasma membrane fraction; ( D , E ) Co-immunoprecipitation of FKBP8 and CLC-1 in skeletal muscle. Muscle lysates were subject to immunoprecipitation ( IP ) with the anti-CLC-1 ( D ) or anti-FKBP8 ( E ) antibody, followed by immunoblotting with the indicated antibodies. Rabbit IgG was employed to verify the specificity of immunoprecipitation. Corresponding expression levels of CLC-1 and FKBP8 in the lysates are shown in the Input lane. Input represents about 10% of the total protein used for immunoprecipitation. h.c. : IgG heavy chain.

    Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using rabbit anti-Aha1 (1:2500; Thermo Scientific, Waltham, MA, USA), mouse anti-cadherin (1:1000; Abcam, Cambridge, MA, USA), mouse anti-calnexin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CLC-1 (1:1000; Alomone, Jerusalem, Israel), rabbit anti-Flag (1:5000; Sigma-Aldrich), rabbit anti-FKBP8 (1:5000; GeneTex, Hsinchu, Taiwan), rabbit anti-GAPDH (1:8000; GeneTex), rat anti-HA (1:5000; Roche Applied Science), rabbit anti-GFP (1:5000; Abcam), mouse anti-GM130 (1:1000; BD Biosciences, San Jose, CA, USA), rabbit anti-HOP (1:10,000; Abcam), rabbit anti-Hsc70 (1:750; Abcam), rabbit anti- Hsp90β (1:500; Abcam), mouse anti-Myc (clone 9E10), or mouse anti-Na+ -K+-ATPase (1:1000; Thermo Scientific) antibodies.

    Techniques: Fractionation, Isolation, Immunoprecipitation, Expressing

    FKBP8 promotes surface protein stability. ( A , B ) Representative immunoblots showing the time course of surface Flag-CLC-1 protein turn-over in the absence ( A ) or presence ( B ) of Myc-FKBP8 over-expression. Transfected HEK293T cells were subject to the indicated durations of treatment with the Golgi trafficking inhibitor brefeldin A (BFA) (5 μg/mL), followed by surface biotinylation analyses; ( C – E ) Quantification of surface Flag-CLC-1 protein stability in the absence ( black ) or presence ( red ) of Myc-FKBP8 over-expression ( n = 6–8); ( C ) Linear plot of the turn-over kinetics of surface CLC-1 protein densities. Protein density was standardized as the ratio of surface Flag-CLC-1 signal to the cognate total GAPDH signal, followed by normalization to the corresponding control at 0 hr; ( D ) Semi-logarithmic plot of the same data points, followed by linear-regression analyses ( solid lines ); ( E ) Statistical analyses of surface CLC-1 protein half-life. The surface protein turn-over time course determined from each experiment was individually plotted on a semi-logarithmic scale for linear-regression analysis, and independent protein half-life values were pooled together for statistical comparisons. The asterisk denotes a significant difference from the control (*, t -test: p

    Journal: International Journal of Molecular Sciences

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    doi: 10.3390/ijms19123783

    Figure Lengend Snippet: FKBP8 promotes surface protein stability. ( A , B ) Representative immunoblots showing the time course of surface Flag-CLC-1 protein turn-over in the absence ( A ) or presence ( B ) of Myc-FKBP8 over-expression. Transfected HEK293T cells were subject to the indicated durations of treatment with the Golgi trafficking inhibitor brefeldin A (BFA) (5 μg/mL), followed by surface biotinylation analyses; ( C – E ) Quantification of surface Flag-CLC-1 protein stability in the absence ( black ) or presence ( red ) of Myc-FKBP8 over-expression ( n = 6–8); ( C ) Linear plot of the turn-over kinetics of surface CLC-1 protein densities. Protein density was standardized as the ratio of surface Flag-CLC-1 signal to the cognate total GAPDH signal, followed by normalization to the corresponding control at 0 hr; ( D ) Semi-logarithmic plot of the same data points, followed by linear-regression analyses ( solid lines ); ( E ) Statistical analyses of surface CLC-1 protein half-life. The surface protein turn-over time course determined from each experiment was individually plotted on a semi-logarithmic scale for linear-regression analysis, and independent protein half-life values were pooled together for statistical comparisons. The asterisk denotes a significant difference from the control (*, t -test: p

    Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using rabbit anti-Aha1 (1:2500; Thermo Scientific, Waltham, MA, USA), mouse anti-cadherin (1:1000; Abcam, Cambridge, MA, USA), mouse anti-calnexin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CLC-1 (1:1000; Alomone, Jerusalem, Israel), rabbit anti-Flag (1:5000; Sigma-Aldrich), rabbit anti-FKBP8 (1:5000; GeneTex, Hsinchu, Taiwan), rabbit anti-GAPDH (1:8000; GeneTex), rat anti-HA (1:5000; Roche Applied Science), rabbit anti-GFP (1:5000; Abcam), mouse anti-GM130 (1:1000; BD Biosciences, San Jose, CA, USA), rabbit anti-HOP (1:10,000; Abcam), rabbit anti-Hsc70 (1:750; Abcam), rabbit anti- Hsp90β (1:500; Abcam), mouse anti-Myc (clone 9E10), or mouse anti-Na+ -K+-ATPase (1:1000; Thermo Scientific) antibodies.

    Techniques: Western Blot, Over Expression, Transfection