Figure 2. Bacterial ligands promote K6a... | Rockefeller University Press

Figure 2.

$Figure 2. Bacterial ligands promote K6a solubility and production of KAMPs. (A–C) Immunoblot analysis of NP-40–soluble and -insoluble lysate fractions of hTCEpi organotypic culture treated with vehicle control or flagellin (FliC; 0.5 µg/ml; A), LPS (1 µg/ml; B), or LTA (1 µg/ml; C) for 16 h. For cells treated with LPS, a 2-h pretreatment with IFN-γ (250 ng/ml) was used to induce LPS responsiveness. Cytosolic K6a (60 kD) in the NP-40–soluble lysate fractions (10 µg total protein) and filamentous K6a (60 kD) in the insoluble fractions (1 µg total protein) were resolved in 12% Bis-Tris gels followed by immunoblotting. Same soluble lysate fractions (40 µg total protein) were also resolved in 16% tricine gel and immunoblotted for KAMPs (∼3 kD). Actin (42 kD) was used as a loading control. Quantification of band intensity was performed with Bio-Rad Laboratories Image Lab software and normalized with actin. Means ± SEM are shown. n = 4–8 independent experiments. **, P < 0.01; ***, P < 0.001. (D) Quantitative RT-PCR assessment of K6a gene expression in hTCEpi cells treated with vehicle control, flagellin (FliC; 0.5 µg/ml), LPS (1 µg/ml), or LTA (1 µg/ml) for 3 and 6 h. K6a gene expression was normalized to actin. Compared with control cells, K6a gene expression in treated cells was unaffected under the indicated conditions (P > 0.05). Means ± SD are shown. Experiments were performed three times.$

Bacterial ligands promote K6a solubility and production of KAMPs. (A–C) Immunoblot analysis of NP-40–soluble and -insoluble lysate fractions of hTCEpi organotypic culture treated with vehicle control or flagellin (FliC; 0.5 µg/ml; A), LPS (1 µg/ml; B), or LTA (1 µg/ml; C) for 16 h. For cells treated with LPS, a 2-h pretreatment with IFN-γ (250 ng/ml) was used to induce LPS responsiveness. Cytosolic K6a (60 kD) in the NP-40–soluble lysate fractions (10 µg total protein) and filamentous K6a (60 kD) in the insoluble fractions (1 µg total protein) were resolved in 12% Bis-Tris gels followed by immunoblotting. Same soluble lysate fractions (40 µg total protein) were also resolved in 16% tricine gel and immunoblotted for KAMPs (∼3 kD). Actin (42 kD) was used as a loading control. Quantification of band intensity was performed with Bio-Rad Laboratories Image Lab software and normalized with actin. Means ± SEM are shown. n = 4–8 independent experiments. **, P < 0.01; ***, P < 0.001. (D) Quantitative RT-PCR assessment of K6a gene expression in hTCEpi cells treated with vehicle control, flagellin (FliC; 0.5 µg/ml), LPS (1 µg/ml), or LTA (1 µg/ml) for 3 and 6 h. K6a gene expression was normalized to actin. Compared with control cells, K6a gene expression in treated cells was unaffected under the indicated conditions (P > 0.05). Means ± SD are shown. Experiments were performed three times.

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