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PNAS 104 (1): 134-138

Copyright © 2007 by the National Academy of Sciences.

Bcl10 and Malt1 control lysophosphatidic acid-induced NF-{kappa}B activation and cytokine production

Stefanie Klemm*, Stephanie Zimmermann*, Christian Peschel*, Tak W. Mak{dagger},{ddagger}, and Jürgen Ruland*,{ddagger}

*Third Medical Department, Technical University of Munich, Klinikum Rechts der Isar, Ismaninger Strasse 22, 81675 Munich, Germany; and {dagger}The Campbell Family Institute for Breast Cancer Research and Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, ON, Canada M5G 2C1


Figure 1
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Fig. 1. Bcl10 regulates LPA-induced NF-{kappa}B activation. (A and B) Bcl10+/– or Bcl10–/– MEFs were preincubated with cycloheximide and then stimulated with 10 µM LPA (A) or 10 ng/ml TNF-{alpha} (B) for the indicated times. Cell extracts were separated on 10% polyacrylamide gels, subjected to immunoblotting with anti-I{kappa}B-{alpha} antibody, and reprobed with anti-beta-actin antibody to show equal loading. Experiments were repeated five times. (C and D) Bcl10+/– or Bcl10–/– MEFs were stimulated with LPA (C) or TNF-{alpha} (D) as described above; nuclear extracts were prepared and incubated with radiolabeled oligonucleotides specific for NF-{kappa}B or NF-Y as a loading control. Results are representative of three independent experiments.

 

Figure 2
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Fig. 2. Retroviral reconstitution of Bcl10 gene-deficient MEFs rescues I{kappa}B-{alpha} degradation in response to LPA stimulation. (A) Bcl10-deficient MEFs were infected with the retroviral vector pBABE-puro encoding full-length Bcl10 (pBABE-puro-Bcl10) or an empty vector (pBABE-puro) as a negative control. Cell extracts were prepared and subjected to immunoblotting with anti-Bcl10-antibody and anti-beta-actin antibody as a loading control. (B) Bcl10-deficient MEFs retrovirally infected with pBABE-puro or pBABE-puro-Bcl10 were stimulated with 10 µM LPA as described above, and cell extracts were subjected to immunoblotting by using antibodies against I{kappa}B-{alpha} and beta-actin. Experiments were repeated three times.

 

Figure 3
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Fig. 3. Normal LPA-induced MAPK and Akt activation in Bcl10-deficient MEFs. Bcl10+/– or Bcl10–/– MEFs were stimulated with 10 µM LPA as indicated. Cell extracts were separated on polyacrylamide gels and immunoblotted with antibodies against phospho-p38, phospho-p44/p42 (phospho-Erk), phospho-Jnk, or phospho-Akt. After stripping, membranes were reprobed with antibodies against p44/p42 (Erk) or beta-actin to control for equal loading. Results are representative of five independent experiments.

 

Figure 4
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Fig. 4. PKC, not Akt, regulates LPA-induced NF-{kappa}B activation. Bcl10+/– or Bcl10–/– MEFs were treated for 1 h with the PKC inhibitor rottlerin (Ro, 3 µM) (A) or the PI3K inhibitor wortmannin (WM, 1 µM) (B) before stimulation with 10 µM LPA for the indicated times. Cell extracts were subjected to immunoblotting with antibodies against I{kappa}B-{alpha}, Bcl10, phospho-Akt, or beta-actin as a loading control. Experiments were repeated independently three times.

 

Figure 5
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Fig. 5. Malt1 regulates LPA-induced NF-{kappa}B activation but not MAPK or Akt activation. (A and B) Malt1+/+, Malt1+/–, or Malt1–/– MEFs were preincubated with cycloheximide and then stimulated with 10 µM LPA (A) or 10 ng/ml TNF-{alpha} (B) for the indicated times. Total extracts were prepared and subjected to immunoblotting with an antibody against I{kappa}B-{alpha} and reprobed with anti-beta-actin antibody for protein loading control. (C and D) Malt1+/+, Malt1+/–, or Malt1–/– MEFs were stimulated with LPA (C) or TNF-{alpha} (D) as described above; nuclear extracts were prepared and incubated with radiolabeled oligonucleotides against NF-{kappa}B or NF-Y as a loading control. (E) Malt1+/– or Malt1–/– MEFs were stimulated with 10 µM LPA as indicated, and cell extracts were immunoblotted with antibodies against phospho-p38, phospho-p44/p42 (phospho-Erk), phospho-Jnk, or phospho-Akt. After stripping, membranes were reprobed with antibodies against p44/p42 (Erk) as a loading control. Results are representative of four different experiments.

 

Figure 6
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Fig. 6. Impaired IL-6 production in Bc10-deficient MEFs. Bcl10+/– or Bcl10–/– MEFs were stimulated with 10 µM LPA for the indicated times. Supernatants were collected, and IL-6 concentrations were determined by ELISA. Data are given as means ± SEM and are representative of three independent experiments.

 


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