Figure 5. The UPS degrades soluble K6a... | Rockefeller University Press

Figure 5.

$Figure 5. The UPS degrades soluble K6a to generate KAMPs. (A) Effects of proteasome inhibition to the cell viability of hTCEpi organotypic culture. Cells pretreated with 0, 50, 100, or 200 nM of epoxomicin for 2 h followed by cotreatment with the same dosage of epoxomicin and LTA (1 µg/ml) for 16 h were incubated with MTT substrate solution or proteasome-Glo cell-based reagent. Cell viability across all epoxomicin concentrations was maintained ∼80–100% in contrast with a dose-dependent decrease in proteasome activities to as low as 20% at the highest concentration of epoxomicin used. Means ± SD (n = 4) are shown. (B) Effect of proteasome inhibition on the level of polyubiquitinated K6a. hTCEpi organotypic culture were treated with DMSO (lanes 1, 3, and 5) or epoxomicin (200 nM; lanes 2, 4, and 6) for 2 h followed by continual treatment with DMSO (vehicle control) or epoxomicin in the absence (lanes 1, 2, and 5) or presence (lanes 3, 4, and 6) of LTA (1 µg/ml) for 18 h. Cells were harvested and disrupted by RIPA lysis buffer, and lysates were immunoprecipitated (IP) with anti-K6a antiserum (lanes 1–4) or preimmune serum (lanes 5 and 6). Ubiquitinated K6a and total K6a were immunoblotted (IB) with antiubiquitin antibody (topmost panel) and anti-K6a antiserum (second panel) respectively. Epoxomicin promotes the accumulation of polyubiquitinated K6a (lanes 2 and 4). (C) Cytosolic K6a was immunoprecipitated from hTCEpi organotypic culture treated with various bacterial ligands in the presence or absence of epoxomicin. LC-MS analysis identified three different ubiquitination sites on K6a. (D) Effect of CRL inhibitor MLN4924 on the level of polyubiquitinated K6a. hTCEpi cells were pretreated with MLN4924 (200 nM) for 1 h followed by DMSO (lanes 1–3) or epoxomicin (lanes 4–6) treatment for 18 h. Cell lysates were immunoprecipitated with preimmune serum (lanes 1 and 4) or anti-K6a antiserum (lanes 2 and 3 and lanes 5 and 6). Note the decrease of polyubiquitinated K6a level when CRL was inhibited. (E) Effect of proteasome inhibition on the level of KAMPs. hTCEpi cells were treated with DMSO (lane 1) or epoxomicin (200 nM; lane 2) for 2 h followed by continual treatment with DMSO and epoxomicin in the presence of LTA (1 µg/ml) for 16 h before cell harvest and disruption by NP-40 lysis buffer to collect cytosolic fractions. The level of KAMPs decreased in the presence of epoxomicin. (F) Trypsin-like proteasome activity of hTCEpi cells was unaffected by bacterial ligand challenge (means ± SD). Experiments were performed at least three times. RLU, relative light unit.$

The UPS degrades soluble K6a to generate KAMPs. (A) Effects of proteasome inhibition to the cell viability of hTCEpi organotypic culture. Cells pretreated with 0, 50, 100, or 200 nM of epoxomicin for 2 h followed by cotreatment with the same dosage of epoxomicin and LTA (1 µg/ml) for 16 h were incubated with MTT substrate solution or proteasome-Glo cell-based reagent. Cell viability across all epoxomicin concentrations was maintained ∼80–100% in contrast with a dose-dependent decrease in proteasome activities to as low as 20% at the highest concentration of epoxomicin used. Means ± SD (n = 4) are shown. (B) Effect of proteasome inhibition on the level of polyubiquitinated K6a. hTCEpi organotypic culture were treated with DMSO (lanes 1, 3, and 5) or epoxomicin (200 nM; lanes 2, 4, and 6) for 2 h followed by continual treatment with DMSO (vehicle control) or epoxomicin in the absence (lanes 1, 2, and 5) or presence (lanes 3, 4, and 6) of LTA (1 µg/ml) for 18 h. Cells were harvested and disrupted by RIPA lysis buffer, and lysates were immunoprecipitated (IP) with anti-K6a antiserum (lanes 1–4) or preimmune serum (lanes 5 and 6). Ubiquitinated K6a and total K6a were immunoblotted (IB) with antiubiquitin antibody (topmost panel) and anti-K6a antiserum (second panel) respectively. Epoxomicin promotes the accumulation of polyubiquitinated K6a (lanes 2 and 4). (C) Cytosolic K6a was immunoprecipitated from hTCEpi organotypic culture treated with various bacterial ligands in the presence or absence of epoxomicin. LC-MS analysis identified three different ubiquitination sites on K6a. (D) Effect of CRL inhibitor MLN4924 on the level of polyubiquitinated K6a. hTCEpi cells were pretreated with MLN4924 (200 nM) for 1 h followed by DMSO (lanes 1–3) or epoxomicin (lanes 4–6) treatment for 18 h. Cell lysates were immunoprecipitated with preimmune serum (lanes 1 and 4) or anti-K6a antiserum (lanes 2 and 3 and lanes 5 and 6). Note the decrease of polyubiquitinated K6a level when CRL was inhibited. (E) Effect of proteasome inhibition on the level of KAMPs. hTCEpi cells were treated with DMSO (lane 1) or epoxomicin (200 nM; lane 2) for 2 h followed by continual treatment with DMSO and epoxomicin in the presence of LTA (1 µg/ml) for 16 h before cell harvest and disruption by NP-40 lysis buffer to collect cytosolic fractions. The level of KAMPs decreased in the presence of epoxomicin. (F) Trypsin-like proteasome activity of hTCEpi cells was unaffected by bacterial ligand challenge (means ± SD). Experiments were performed at least three times. RLU, relative light unit.

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