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Bio-Rad hamster anti mouse cd81 mab

( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and <t>CD81</t> were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Hamster Anti Mouse Cd81 Mab, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hamster anti mouse cd81 mab - by Bioz Stars, 2026-07

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1) Product Images from "Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages"

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073706

( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Figure Legend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Techniques Used: Cell Culture, Western Blot, Expressing, Flow Cytometry

( A ) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). ( C ) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of CD9 and CD81 were examined by reverse transcription PCR. GAPDH is an internal loading control. ( D ) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of CD9 and CD81 were examined by real-time PCR. Data shown are from one representative of three similar experiments. ( E ) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. ( F ) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.

Figure Legend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). ( C ) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of CD9 and CD81 were examined by reverse transcription PCR. GAPDH is an internal loading control. ( D ) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of CD9 and CD81 were examined by real-time PCR. Data shown are from one representative of three similar experiments. ( E ) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. ( F ) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.

Techniques Used: Cell Culture, Western Blot, Reverse Transcription, Control, Real-time Polymerase Chain Reaction

$( A ) RAW264.7 cells were stimulated with 0.1 µg/ml LPS and, after the indicated times, the cells were lysed and protein levels were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured for 24 h in the absence (-) or presence of 5 µM fluvastatin (Fluv) or simvastatin (Simv) (+) and stimulated for 2 h with 1 µg/ml LPS (+). Proteins in whole-cell lysate (WCL) and CD14 protein in immunoprecipitates (IP) with anti-TLR4 Ab were immunoblotted (IB). ( C ) RAW264.7 cells were treated as in B . Lysates of untreated (C, control) cultures or LPS-stimulated cultures in the absence (L) or presence of fluvastatin (FL) or simvastatin (SL) were fractionated by sucrose density gradients, and protein distributions were visualized by immunoblotting. The intensities of blots were quantified by densitometry, and percentages of density units of light membrane (LM) fractions are displayed to the right of the blots. Data shown are from one representative of three similar experiments. ( D ) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb. ( E ) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb from pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10). In the presence of statins, more CD14/CD9 complexes were formed in dense fractions (arrowheads).$
Figure Legend Snippet: ( A ) RAW264.7 cells were stimulated with 0.1 µg/ml LPS and, after the indicated times, the cells were lysed and protein levels were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured for 24 h in the absence (-) or presence of 5 µM fluvastatin (Fluv) or simvastatin (Simv) (+) and stimulated for 2 h with 1 µg/ml LPS (+). Proteins in whole-cell lysate (WCL) and CD14 protein in immunoprecipitates (IP) with anti-TLR4 Ab were immunoblotted (IB). ( C ) RAW264.7 cells were treated as in B . Lysates of untreated (C, control) cultures or LPS-stimulated cultures in the absence (L) or presence of fluvastatin (FL) or simvastatin (SL) were fractionated by sucrose density gradients, and protein distributions were visualized by immunoblotting. The intensities of blots were quantified by densitometry, and percentages of density units of light membrane (LM) fractions are displayed to the right of the blots. Data shown are from one representative of three similar experiments. ( D ) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb. ( E ) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb from pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10). In the presence of statins, more CD14/CD9 complexes were formed in dense fractions (arrowheads).

Techniques Used: Western Blot, Cell Culture, Control, Membrane

( A ) BMDMs from WT mice were cultured for 24 h in the absence (-) or presence of 3 µM fluvastatin (Fluv) (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) BMDMs from WT and CD9 KO mice were cultured in the absence or presence of the indicated concentrations of fluvastatin, and stimulated for 18 h with 10 µg/ml LPS (+). Activities of MMP-9 in culture supernatants were analyzed by gelatin zymography. ( C ) BMDMs from WT and CD9 KO mice were cultured in the absence (vehicle) or presence of 10 µM fluvastatin or simvastatin (Simv), and unstimulated (-) or stimulated for 18 h with 1 µg/ml LPS (+). Concentrations of TNF-α in culture supernatants were measured by ELISA. Each bar represents the mean ± SEM. ⋆ P < 0.05; ⋆ ⋆ P < 0.01.

Figure Legend Snippet: ( A ) BMDMs from WT mice were cultured for 24 h in the absence (-) or presence of 3 µM fluvastatin (Fluv) (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) BMDMs from WT and CD9 KO mice were cultured in the absence or presence of the indicated concentrations of fluvastatin, and stimulated for 18 h with 10 µg/ml LPS (+). Activities of MMP-9 in culture supernatants were analyzed by gelatin zymography. ( C ) BMDMs from WT and CD9 KO mice were cultured in the absence (vehicle) or presence of 10 µM fluvastatin or simvastatin (Simv), and unstimulated (-) or stimulated for 18 h with 1 µg/ml LPS (+). Concentrations of TNF-α in culture supernatants were measured by ELISA. Each bar represents the mean ± SEM. ⋆ P < 0.05; ⋆ ⋆ P < 0.01.

Techniques Used: Cell Culture, Western Blot, Zymography, Enzyme-linked Immunosorbent Assay

( A ) RAW264.7 cells were untreated (-) or treated for 48 h with 50 ng/ml TSA (+) in the absence (-) or presence of 50 µM theophylline or 0.5 µM fluvastatin (Fluv) (+). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) The mevalonate pathway and inhibitors. n-BP, nitrogenous bisphosphonate. ( C ) RAW264.7 cells were cultured for 24 h in the presence of indicated concentrations of fluvastatin, simvastatin (Simv), zoledronate (Zol), or risedronate (Ris). Levels of CD9 and CD81 were examined by immunoblotting. ( D ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) or presence of mevalonate (Mev), farnesyl pyrophosphate (FPP), squalene (Squ), or geranylgeranyl pyrophosphate (GGPP). Although the actin level in the GGPP lane appears to be lower, an equal amount of protein was loaded. ( E ) RAW264.7 cells were cultured for 24 h in the absence (V) or presence of fluvastatin, zoledronate, farnesyl transferase inhibitor (FTI), or geranylgeranyl transferase inhibitor (GGTI). ( F ) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. ( G ) RAW264.7 cells were untreated (-) or treated with zoledronate (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. ( H ) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP and stimulated for 15 min with 0.1 µg/ml LPS (+). The cells were lysed, and levels of IκBα were examined by immunoblotting. ( I ) RAW264.7 cells were cultured for 24 h in the indicated concentrations of HA1077. Levels of CD9 and CD81 were examined by immunoblotting.

Figure Legend Snippet: ( A ) RAW264.7 cells were untreated (-) or treated for 48 h with 50 ng/ml TSA (+) in the absence (-) or presence of 50 µM theophylline or 0.5 µM fluvastatin (Fluv) (+). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) The mevalonate pathway and inhibitors. n-BP, nitrogenous bisphosphonate. ( C ) RAW264.7 cells were cultured for 24 h in the presence of indicated concentrations of fluvastatin, simvastatin (Simv), zoledronate (Zol), or risedronate (Ris). Levels of CD9 and CD81 were examined by immunoblotting. ( D ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) or presence of mevalonate (Mev), farnesyl pyrophosphate (FPP), squalene (Squ), or geranylgeranyl pyrophosphate (GGPP). Although the actin level in the GGPP lane appears to be lower, an equal amount of protein was loaded. ( E ) RAW264.7 cells were cultured for 24 h in the absence (V) or presence of fluvastatin, zoledronate, farnesyl transferase inhibitor (FTI), or geranylgeranyl transferase inhibitor (GGTI). ( F ) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. ( G ) RAW264.7 cells were untreated (-) or treated with zoledronate (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. ( H ) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP and stimulated for 15 min with 0.1 µg/ml LPS (+). The cells were lysed, and levels of IκBα were examined by immunoblotting. ( I ) RAW264.7 cells were cultured for 24 h in the indicated concentrations of HA1077. Levels of CD9 and CD81 were examined by immunoblotting.

Techniques Used: Western Blot, Cell Culture

Image Search Results

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). ( C ) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of CD9 and CD81 were examined by reverse transcription PCR. GAPDH is an internal loading control. ( D ) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of CD9 and CD81 were examined by real-time PCR. Data shown are from one representative of three similar experiments. ( E ) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. ( F ) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.

Techniques: Cell Culture, Western Blot, Reverse Transcription, Control, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were stimulated with 0.1 µg/ml LPS and, after the indicated times, the cells were lysed and protein levels were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured for 24 h in the absence (-) or presence of 5 µM fluvastatin (Fluv) or simvastatin (Simv) (+) and stimulated for 2 h with 1 µg/ml LPS (+). Proteins in whole-cell lysate (WCL) and CD14 protein in immunoprecipitates (IP) with anti-TLR4 Ab were immunoblotted (IB). ( C ) RAW264.7 cells were treated as in B . Lysates of untreated (C, control) cultures or LPS-stimulated cultures in the absence (L) or presence of fluvastatin (FL) or simvastatin (SL) were fractionated by sucrose density gradients, and protein distributions were visualized by immunoblotting. The intensities of blots were quantified by densitometry, and percentages of density units of light membrane (LM) fractions are displayed to the right of the blots. Data shown are from one representative of three similar experiments. ( D ) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb. ( E ) Immunoblots of CD9 and CD81 proteins in whole-cell lysates and in immunoprecipitates with control IgG or anti-CD14 mAb from pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10). In the presence of statins, more CD14/CD9 complexes were formed in dense fractions (arrowheads).

Techniques: Western Blot, Cell Culture, Control, Membrane

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) BMDMs from WT mice were cultured for 24 h in the absence (-) or presence of 3 µM fluvastatin (Fluv) (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) BMDMs from WT and CD9 KO mice were cultured in the absence or presence of the indicated concentrations of fluvastatin, and stimulated for 18 h with 10 µg/ml LPS (+). Activities of MMP-9 in culture supernatants were analyzed by gelatin zymography. ( C ) BMDMs from WT and CD9 KO mice were cultured in the absence (vehicle) or presence of 10 µM fluvastatin or simvastatin (Simv), and unstimulated (-) or stimulated for 18 h with 1 µg/ml LPS (+). Concentrations of TNF-α in culture supernatants were measured by ELISA. Each bar represents the mean ± SEM. ⋆ P < 0.05; ⋆ ⋆ P < 0.01.

Techniques: Cell Culture, Western Blot, Zymography, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were untreated (-) or treated for 48 h with 50 ng/ml TSA (+) in the absence (-) or presence of 50 µM theophylline or 0.5 µM fluvastatin (Fluv) (+). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) The mevalonate pathway and inhibitors. n-BP, nitrogenous bisphosphonate. ( C ) RAW264.7 cells were cultured for 24 h in the presence of indicated concentrations of fluvastatin, simvastatin (Simv), zoledronate (Zol), or risedronate (Ris). Levels of CD9 and CD81 were examined by immunoblotting. ( D ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) or presence of mevalonate (Mev), farnesyl pyrophosphate (FPP), squalene (Squ), or geranylgeranyl pyrophosphate (GGPP). Although the actin level in the GGPP lane appears to be lower, an equal amount of protein was loaded. ( E ) RAW264.7 cells were cultured for 24 h in the absence (V) or presence of fluvastatin, zoledronate, farnesyl transferase inhibitor (FTI), or geranylgeranyl transferase inhibitor (GGTI). ( F ) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. ( G ) RAW264.7 cells were untreated (-) or treated with zoledronate (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP. ( H ) RAW264.7 cells were untreated (-) or treated with fluvastatin (+) in the absence (V) or presence of mevalonate, FPP, squalene, or GGPP and stimulated for 15 min with 0.1 µg/ml LPS (+). The cells were lysed, and levels of IκBα were examined by immunoblotting. ( I ) RAW264.7 cells were cultured for 24 h in the indicated concentrations of HA1077. Levels of CD9 and CD81 were examined by immunoblotting.

Techniques: Western Blot, Cell Culture

FIGURE 1. Expression of macrophage CD9 and CD81 in the absence or presence of LPS. A, RAW264.7 macrophages were serum-starved and cul- tured in the absence () or presence () of 0.1 g/ml LPS. After the indicated hours, the cells were photographed. Bar, 50 m. B, After the indicated hours, lysates of RAW264.7 cells were electrophoresed on SDS- PAGE and transferred to an Immobilon-P membrane. CD9 and CD81 were immunoblotted with anti-CD9 and anti-CD81 mAbs. Anti-actin blots show comparable amounts of protein loaded in each lane (lower panels). The intensity of blots was quantiﬁed by densitometry (upper panels). C, Mouse BMDMs were serum-starved and cultured in the absence or presence of 1 g/ml LPS. After the indicated hours, the cells were lysed and protein levels of CD9 and CD81 were examined by immunoblotting (lower pan- els). The intensity of blots was quantiﬁed by densitometry (upper panels). Blots shown in the lower panels are from one representative of three in- dependent experiments and each data point in the upper panels represents the mean SEM. , p 0.05 vs 0 h; , p 0.01 vs 0 h. Asterisks for CD9 and CD81 are shown above and below symbols, respectively.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tetraspanin CD9 negatively regulates lipopolysaccharide-induced macrophage activation and lung inflammation.

doi: 10.4049/jimmunol.0802797

Figure Lengend Snippet: FIGURE 1. Expression of macrophage CD9 and CD81 in the absence or presence of LPS. A, RAW264.7 macrophages were serum-starved and cul- tured in the absence () or presence () of 0.1 g/ml LPS. After the indicated hours, the cells were photographed. Bar, 50 m. B, After the indicated hours, lysates of RAW264.7 cells were electrophoresed on SDS- PAGE and transferred to an Immobilon-P membrane. CD9 and CD81 were immunoblotted with anti-CD9 and anti-CD81 mAbs. Anti-actin blots show comparable amounts of protein loaded in each lane (lower panels). The intensity of blots was quantiﬁed by densitometry (upper panels). C, Mouse BMDMs were serum-starved and cultured in the absence or presence of 1 g/ml LPS. After the indicated hours, the cells were lysed and protein levels of CD9 and CD81 were examined by immunoblotting (lower pan- els). The intensity of blots was quantiﬁed by densitometry (upper panels). Blots shown in the lower panels are from one representative of three in- dependent experiments and each data point in the upper panels represents the mean SEM. , p 0.05 vs 0 h; , p 0.01 vs 0 h. Asterisks for CD9 and CD81 are shown above and below symbols, respectively.

Article Snippet: Rat anti-mouse CD9 mAb (KMC8) and hamster anti-mouse CD81 mAb (Eat2) were purchased from BD Biosciences and U.K.-Serotec, respectively.

Techniques: Expressing, SDS Page, Membrane, Cell Culture, Western Blot

$FIGURE 4. The expression of CD14, complex formation of CD14 and TLR4, and their localization into the lipid raft are enhanced in CD9 KO BMDMs. A, BMDMs from WT and CD9 KO mice were stimulated with 1 g/ml LPS. After the indicated hours, cell lysates were electrophoresed on SDS-PAGE, transferred to a membrane, probed with anti-CD14 and anti-TLR4 Abs. Anti-actin blots show comparable amounts of protein loaded in each lane. B, WT and CD9 KO BMDMs were cultured in the absence () or presence () of LPS for 20 h. CD14 protein in whole cell lysates (WCL) and in immuno- precipitates with anti-TLR4 mAb (MTS510) was immunoblotted with biotinylated anti-CD14 Ab after SDS-PAGE. Anti-actin blots show comparable amounts of protein loaded in each lane. C, Cell lysates from WT and CD9 KO BMDMs cultured in the absence () or presence () of LPS for 2 h were centrifuged in sucrose density gradients. Fractions were collected from the top of the gradient and separated by SDS-PAGE. Protein distribution in the fractions was visualized by immunoblotting with anti-CD14, anti-TLR4, anti-CD9, anti-CD81, anti-CD45, and anti-ﬂotillin-1 Abs. GM1 ganglioside was detected with HRP-conjugated cholera toxin by dot blot. The intensity of blots was quantiﬁed by densitometry. Percentage of density units of light membrane (LM) fractions (4 plus 5) were calculated and shown on the right of blots. D, Pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10) from the sucrose gradients of WT cell lysate were subjected to imunoprecipiation with anti-CD9 mAb. After separation of the immunoprecipitates on SDS-PAGE, CD14 and CD9 were immunoblotted with biotinylated anti-CD14 Ab and anti-CD9 mAb, respectively. Data shown are from one representative of three similar experiments.$

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tetraspanin CD9 negatively regulates lipopolysaccharide-induced macrophage activation and lung inflammation.

doi: 10.4049/jimmunol.0802797

Figure Lengend Snippet: FIGURE 4. The expression of CD14, complex formation of CD14 and TLR4, and their localization into the lipid raft are enhanced in CD9 KO BMDMs. A, BMDMs from WT and CD9 KO mice were stimulated with 1 g/ml LPS. After the indicated hours, cell lysates were electrophoresed on SDS-PAGE, transferred to a membrane, probed with anti-CD14 and anti-TLR4 Abs. Anti-actin blots show comparable amounts of protein loaded in each lane. B, WT and CD9 KO BMDMs were cultured in the absence () or presence () of LPS for 20 h. CD14 protein in whole cell lysates (WCL) and in immuno- precipitates with anti-TLR4 mAb (MTS510) was immunoblotted with biotinylated anti-CD14 Ab after SDS-PAGE. Anti-actin blots show comparable amounts of protein loaded in each lane. C, Cell lysates from WT and CD9 KO BMDMs cultured in the absence () or presence () of LPS for 2 h were centrifuged in sucrose density gradients. Fractions were collected from the top of the gradient and separated by SDS-PAGE. Protein distribution in the fractions was visualized by immunoblotting with anti-CD14, anti-TLR4, anti-CD9, anti-CD81, anti-CD45, and anti-ﬂotillin-1 Abs. GM1 ganglioside was detected with HRP-conjugated cholera toxin by dot blot. The intensity of blots was quantiﬁed by densitometry. Percentage of density units of light membrane (LM) fractions (4 plus 5) were calculated and shown on the right of blots. D, Pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10) from the sucrose gradients of WT cell lysate were subjected to imunoprecipiation with anti-CD9 mAb. After separation of the immunoprecipitates on SDS-PAGE, CD14 and CD9 were immunoblotted with biotinylated anti-CD14 Ab and anti-CD9 mAb, respectively. Data shown are from one representative of three similar experiments.

Article Snippet: Rat anti-mouse CD9 mAb (KMC8) and hamster anti-mouse CD81 mAb (Eat2) were purchased from BD Biosciences and U.K.-Serotec, respectively.

Techniques: Expressing, SDS Page, Membrane, Cell Culture, Western Blot, Dot Blot

FIGURE 1. TSA or CSE down-regulates CD9 and CD81 while up-regulating MMPs in RAW264.7 macrophages. A, RAW264.7 cells were cultured in the absence or presence of TSA, theophylline, and dexamethasone for 48 h. Expressions of CD9, CD81, and integrin 1 were examined by immunoblot- ting using whole cell lysates. Anti-actin blots confirm equal amounts of pro- tein loaded in each lane. B, RAW264.7 was cultured in the absence (filled histograms) or presence (open histograms) of TSA. Surface expressions of CD9, CD81, and integrin 1 were analyzed by flow cytometry. C, A549 alveolar epithelial cells were cultured in the absence or presence of TSA. CD9 and CD81 were immunoblotted. D, RAW264.7 was cultured in the absence or presence of 0.1% CSE for 48 h. CD9, CD81, and integrin 1 were immuno- blotted. E, RAW264.7 was cultured in the absence or presence of TSA. Expres- sions of MMP-2, MMP-9, and MMP-12 were analyzed by RT-PCR. -Actin amplification was internal control.

Journal: Journal of Biological Chemistry

Article Title: Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

doi: 10.1074/jbc.m801902200

Figure Lengend Snippet: FIGURE 1. TSA or CSE down-regulates CD9 and CD81 while up-regulating MMPs in RAW264.7 macrophages. A, RAW264.7 cells were cultured in the absence or presence of TSA, theophylline, and dexamethasone for 48 h. Expressions of CD9, CD81, and integrin 1 were examined by immunoblot- ting using whole cell lysates. Anti-actin blots confirm equal amounts of pro- tein loaded in each lane. B, RAW264.7 was cultured in the absence (filled histograms) or presence (open histograms) of TSA. Surface expressions of CD9, CD81, and integrin 1 were analyzed by flow cytometry. C, A549 alveolar epithelial cells were cultured in the absence or presence of TSA. CD9 and CD81 were immunoblotted. D, RAW264.7 was cultured in the absence or presence of 0.1% CSE for 48 h. CD9, CD81, and integrin 1 were immuno- blotted. E, RAW264.7 was cultured in the absence or presence of TSA. Expres- sions of MMP-2, MMP-9, and MMP-12 were analyzed by RT-PCR. -Actin amplification was internal control.

Article Snippet: Cell lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with rat anti-mouse CD9 (KMC8) and integrin 1 (KMI6) mAbs (BD Biosciences) and hamster antimouse CD81 mAb (Eat2; AbD Serotec, Oxford, UK).

Techniques: Cell Culture, Western Blot, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Amplification, Control

FIGURE 2. mAbs or siRNA to CD9 and CD81 enhance MMP production and suppress motility of RAW264.7 cells. A, RAW264.7 cells were cultured for 24 h in the absence () or presence of KMC8 plus 2F7 (). mRNA was extracted, and RT-PCR was performed for expressions of MMPs and TIMPs. B, RAW264.7 cells were cultured for 24 h in the absence () or presence of the indicated mAbs. MMP-9 activity in culture supernatant was examined by gel- atin zymography (lower) and quantified by densitometry (upper). The gelatin zymography is from one representative of three similar experiments. C, RAW264.7 was transfected with siRNAs against CD9 or CD81. Decrease in CD9 or CD81 was shown in immunoblotting (upper). MMP-9 activity in super- natants of 24-hour culture was examined by gelatin zymography (lower). D, RAW264.7 cells were applied into the upper chamber of FN-precoated Transwells in the absence () or presence of the indicated mAbs. DMEM containing 10% FBS was added to the lower chamber. After 4 h, cells migrat- ing to the lower surface of the membrane were counted after Diff-Quick stain. Bars represent the mean S.E. *, p 0.05 versus IgG; **, p 0.05 versus KMC8. KMC8, anti-CD9; 2F7, anti-CD81; HM1-1, anti-integrin 1.

Journal: Journal of Biological Chemistry

Article Title: Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

doi: 10.1074/jbc.m801902200

Figure Lengend Snippet: FIGURE 2. mAbs or siRNA to CD9 and CD81 enhance MMP production and suppress motility of RAW264.7 cells. A, RAW264.7 cells were cultured for 24 h in the absence () or presence of KMC8 plus 2F7 (). mRNA was extracted, and RT-PCR was performed for expressions of MMPs and TIMPs. B, RAW264.7 cells were cultured for 24 h in the absence () or presence of the indicated mAbs. MMP-9 activity in culture supernatant was examined by gel- atin zymography (lower) and quantified by densitometry (upper). The gelatin zymography is from one representative of three similar experiments. C, RAW264.7 was transfected with siRNAs against CD9 or CD81. Decrease in CD9 or CD81 was shown in immunoblotting (upper). MMP-9 activity in super- natants of 24-hour culture was examined by gelatin zymography (lower). D, RAW264.7 cells were applied into the upper chamber of FN-precoated Transwells in the absence () or presence of the indicated mAbs. DMEM containing 10% FBS was added to the lower chamber. After 4 h, cells migrat- ing to the lower surface of the membrane were counted after Diff-Quick stain. Bars represent the mean S.E. *, p 0.05 versus IgG; **, p 0.05 versus KMC8. KMC8, anti-CD9; 2F7, anti-CD81; HM1-1, anti-integrin 1.

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Zymography, Transfection, Western Blot, Membrane, Diff-Quik, Staining

FIGURE 3. CD9/CD81 DKO mice develop pulmonary emphysema. A, histo- logical lung sections from DKO mice and WT littermates at 3 and 10 weeks of age were stained with hematoxylin and eosin. Bar, 50 m. B, chord length measured from the WT and DKO lungs. C and D, functional tests of the lung. Lung compliance (C) and functional residual capacity (FRC) (D) of mice at 23 weeks of age were measured with a whole body plethysmograph. Values were normalized to body weight. At least three mice were used for each group. Bars represent the mean S.E. *, p 0.05 versus WT.

Journal: Journal of Biological Chemistry

Article Title: Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

doi: 10.1074/jbc.m801902200

Figure Lengend Snippet: FIGURE 3. CD9/CD81 DKO mice develop pulmonary emphysema. A, histo- logical lung sections from DKO mice and WT littermates at 3 and 10 weeks of age were stained with hematoxylin and eosin. Bar, 50 m. B, chord length measured from the WT and DKO lungs. C and D, functional tests of the lung. Lung compliance (C) and functional residual capacity (FRC) (D) of mice at 23 weeks of age were measured with a whole body plethysmograph. Values were normalized to body weight. At least three mice were used for each group. Bars represent the mean S.E. *, p 0.05 versus WT.

Techniques: Staining, Functional Assay

FIGURE 7. CD9/CD81 DKO mice display body weight loss, kyphosis, and osteopenia. A, whole body radiographs of WT and DKO mice at 3 and 30 weeks of age. Bars, 10 mm. B, time course of body weight of WT and DKO littermates. C, toluidine blue stain of longitudinal sections of proximal tibia at 8 weeks of age. Note that cortical bone of the DKO mouse is thinner than that of the WT littermate (arrowheads). Bar, 250 m. D, pQCT images of femoral diaphysis at 8 weeks of age (upper). Mineral densities are shown as different colors according to the standard mineral density gradients. Note the reduc- tion in cortical thickness of the DKO mouse (arrowheads). Total mineral con- tent and strength strain index (SSI) were also determined (lower). Trabecular parameters were not different between WT and DKO mice (data not shown). Values represent the mean S.E. *, p 0.05 versus WT.

Journal: Journal of Biological Chemistry

Article Title: Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

doi: 10.1074/jbc.m801902200

Figure Lengend Snippet: FIGURE 7. CD9/CD81 DKO mice display body weight loss, kyphosis, and osteopenia. A, whole body radiographs of WT and DKO mice at 3 and 30 weeks of age. Bars, 10 mm. B, time course of body weight of WT and DKO littermates. C, toluidine blue stain of longitudinal sections of proximal tibia at 8 weeks of age. Note that cortical bone of the DKO mouse is thinner than that of the WT littermate (arrowheads). Bar, 250 m. D, pQCT images of femoral diaphysis at 8 weeks of age (upper). Mineral densities are shown as different colors according to the standard mineral density gradients. Note the reduc- tion in cortical thickness of the DKO mouse (arrowheads). Total mineral con- tent and strength strain index (SSI) were also determined (lower). Trabecular parameters were not different between WT and DKO mice (data not shown). Values represent the mean S.E. *, p 0.05 versus WT.

Techniques: Staining

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