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1.
Figure 8

Figure 8. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

MD-2 and MD-2C95Y colocalize with TLR4. HEK 293 cells were transiently transfected with human MD-2Flag (lane 1), MD-2Flag plus human TLR4Myc (lane 2), or huMD-2C95YFlag plus TLR4Myc (lane 3). After 48 h cells were harvested, lysed, and immunoprecipitated with anti-Flag agarose. Western blot analysis was performed using an anti-Flag mAb or an anti–c-Myc mAb. Depicted is one representative experiment out of two.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
2.
Figure 3

Figure 3. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

Transfection of the mutant 7.19 with mouse MD-2 confers normal sensitivity to LPS. The parental cell line 3E10 was cotransfected with plasmid pcDNA3 and the pELAM-luc reporter plasmid (black bars), as described in the legend to . Similarly, mutant strain 7.19 was cotransfected with mouse MD-2 and pELAM-luc reporter plasmid (hashed bars). Subsequently, cells were stimulated with medium alone, LPS, or IL-1β for 5 h, and cellular lysates were assayed for luciferase activity. The results shown are mean values of triplicate determinations ±SD and are representative of three independent experiments.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
3.
Figure 7

Figure 7. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

A point mutation at position 95 of the human MD-2 gene abolishes cell activation by LPS. HEK 293 cells were cotransfected with either wild-type human MD-2 plus human TLR4 (hTOLL) or human MD-2C95Y plus TLR4 and the pELAM-luc reporter plasmid. The next day cells were stimulated with medium alone, IL-1β, or LPS at the indicated concentrations for 5 h. Total cellular lysates were assayed for luciferase activity. The results shown are mean values of triplicate determinations ±SD and are representative of three independent experiments.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
4.

Figure 1. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

Mutant CHO-CD14 cells show defects in response to LPS stimulation, but not in response to the cytokines TNF-α and IL-1β. (A) Wild-type 3E10 cells and mutant cell lines 7.7 and 7.19 were stimulated for 15 min with LPS, TNF-α, or IL-1β. Cells were lysed and proteins were resolved by PAGE, electroblotted, and immunoblotted with an Ab specific for phospho-JNK. The blot shown is representative of three independent experiments. (B) Wild-type 3E10 and the mutant cell lines 7.7 and 7.19 were stimulated with LPS or IL-1β at the indicated concentrations for 7.5 h. Supernatants were assayed for bioactive IL-6. The results shown are mean values of triplicate determinations ±SD and are representative of three independent experiments. +, 3E10; ▴, 7.19; X, 7.7.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
5.

Figure 5. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

The mutant MD-2C95Y is transcribed in normal quantities and expressed on the cell surface. (A) Semiquantitative PCR of MD-2 and GAPDH on reverse transcribed mRNA from 3E10 and 7.19 cells. (B) FACS® analysis showing surface expression of wild-type MD-2 and MD-2C95Y in transiently transfected 7.19 cells. Cells were transiently transfected with MD-2 expression plasmids coding for Flag epitope–tagged MD-2. After overnight incubation, cells were detached with 1 mM Na2EDTA, washed, and stained with anti-Flag mAb and FITC-conjugated sheep anti–mouse IgG secondary Ab as described in Materials and Methods. Controls were stained with the secondary Ab only.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
6.

Figure 6. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

Transfection of the nonresponder mutant 7.19 with wild-type CHO MD-2 enables a response to LPS, whereas transfection with MD-2C95Y does not. (A) The mutant strain 7.19 was cotransfected with either wild-type hamster MD-2 or hamster MD-2C95Y plus the pELAM-luc reporter plasmid. The next day cells were stimulated with medium alone, IL-1β, or LPS at the indicated concentrations for 5 h. Total cellular lysates were assayed for luciferase activity. (B) The mutant strain 7.19 was cotransfected with pcDNA3 control plasmid, wild-type hamster MD-2, or hamster MD-2C95Y. The next day cells were stimulated with LPS or IL-1β at the indicated concentrations for 7.5 h and supernatants were assayed for bioactive IL-6. The results shown are mean values of triplicate determinations ±SD and are representative of two independent experiments.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
7.

Figure 4. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

The mutant 7.19 has a point mutation in the gene coding for MD-2. (A) MD-2 was sequenced from 3E10, 7.19 and mutant 7.7 (data not shown); a partial sequence of MD-2 from clone 3E10 and 7.19 is shown in this figure. Mutant 7.19 has a point mutation at position 284 of the coding region resulting in a conversion of the codon for cysteine to tyrosine (codon triplet marked by dashed line). The affected codon corresponds to amino acid position 95 of the protein. (B) Sequence alignment of bp 268–300 from human, mouse, wild-type CHO MD-2 from 3E10 cells, and MD-2 from the mutant 7.19, showing that the codon for cysteine at position 284 in the gene for MD-2, as well as most of the surrounding amino acids, are conserved in a variety of species.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
8.

Figure 2. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

Mouse MD-2 confers LPS signaling when expressed in the LPS nonresponder mutant 7.19, but not on the mutant 7.7. LPS nonresponder mutant cell lines 7.7 (A) and 7.19 (B) were transiently cotransfected with the control plasmid pcDNA3, plasmid coding for human TLR4 (hTOLL), or mouse MD-2, plus the pELAM-luc reporter plasmid as described in Materials and Methods. The next day, cells were stimulated with medium alone, 2.5 ng of IL-1β per milliliter, or 5 μg of LPS per milliliter for 5 h. Total cellular lysates were assayed for luciferase activity. The results shown are mean values of triplicate determinations ±SD and are representative of three independent experiments.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.
9.
Figure 9

Figure 9. From: Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line.

Human wild-type MD-2 but not MD-2C95Y–containing supernatants confer LPS responsiveness on TLR4-transfected cells. HEK 293 cells were transiently cotransfected with either human TLR4 (huTLR4) or empty vector (pcDNA3) plus the pELAM-luc reporter plasmid. The next day, supernatants from separate dishes of HEK 293 cells that had been transiently transfected overnight with either Flag-tagged human wild-type MD-2, human mutant MD-2C95Y, or pcDNA3 were transferred to TLR4- or pcDNA3-transfected cells. These supernatant-treated cells were then stimulated with LPS (100 ng/ml), IL-1β (2.5 ng/ml), or left untreated. After incubation for 5 h, the total cellular lysates were assayed for luciferase activity. The results shown are the mean values of triplicate determinations (±SD) and are representative of three nearly identical independent experiments.

Andra B. Schromm, et al. J Exp Med. 2001 July 2;194(1):79-88.

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