Journal: Cell death and differentiation

Article Title: Identification of PP2A as a crucial regulator of the NF-kappaB feedback loop: its inhibition by UVB turns NF-kappaB into a pro-apoptotic factor.

doi: 10.1038/cdd.2008.98

Figure Lengend Snippet: Figure 4 Failure of IkBa resynthesis is due to continuous inhibitor of kB kinaseb (IKKb) phosphorylation. (a) Cells were stimulated with interleukin-1 (IL-1) or IL-1 þ ultraviolet-B light (UVB). At the indicated time points IkBa transcription was analysed by semiquantitative RT-PCR. (b) Cells were stimulated with IL-1 or co-stimulated with IL-1 þ UVB. MG132 was added 1 h prior to () or 15 min after ( þ ) IL-1/UVB stimulation. Cytosolic IkBa status was determined by WB. (c) Cells were stimulated with IL-1 or IL-1 þ UVB. MG132 was added 1 h prior to () or 15 min after ( þ ) IL-1/UVB stimulation. Specific Ser-Phosphorylation of IkBa was monitored and confirmed by reprobing the stripped membrane with an IkBa antibody. (d) Cells were stimulated with IL-1 or IL-1 þ UVB as indicated. BAY-11-7082 (30 mM) was added 15 min following initial IL-1/ UVB stimulation. Resynthesis of IkBa was detected by WB. (e) Cells were stimulated with IL-1 with or without UVB as annotated and the phosphorylation status of IKKb as well as IkBa degradation determined by WB. (f) Cells stably expressing IKKb-GFP were stimulated with IL-1 or IL-1 þ UVB for 5 min or 2 h. IKKb-GFP was immunoprecipitated and subjected to an in vitro kinase assay with a purified GST-IkBa(5–55) peptide. IkBa, and phospho-IKKb statuses were determined by WB

Article Snippet: For WB analysis cytosolic and nuclear protein extracts were detected with antibodies against IkB-a, P-IkBa-Ser32/36, P-IKKb-Ser177/181, PP2Ac (L35A5, 5A5, 16A6, no. 2038; Cell Signaling), IKKb (10A9B6; Imgenex) and a-tubulin (DM1A; Neomarkers).

Techniques: Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Membrane, Stable Transfection, Expressing, Immunoprecipitation, In Vitro, Kinase Assay

Journal: Cell death and differentiation

Article Title: Identification of PP2A as a crucial regulator of the NF-kappaB feedback loop: its inhibition by UVB turns NF-kappaB into a pro-apoptotic factor.

doi: 10.1038/cdd.2008.98

Figure Lengend Snippet: Figure 6 Loss of PP2Ac results in constitutive activation of inhibitor of kB kinaseb (IKKb) and production of tumour necrosis factor-a (TNF). (a) KB cells were stimulated with interleukin-1 (IL-1) or IL-1 þ ultraviolet-B light (UVB) for 2 h. PP2Ac was immunoprecipitated and co-precipitation of IKKb is shown by WB. (b) Cells were transfected with scrambled- or PP2Ac-siRNA and stimulated with IL-1 and/or calyculin A (5 nM). After 2 h cytosolic IkBa and phospho-IKKb statuses as well as the PP2Ac knockdown were documented by WB. (c) Cells stably expressing IKKb-GFP were transfected with scrambled or PP2Ac-siRNA and stimulated with IL-1 or UVB þ IL-1 for 2 h. IKKb-GFP was immunoprecipitated and subjected to an in vitro kinase assay with a purified GST-IkBa(5–55) peptide. IkBa and phospho-IKKb status were determined by WB. (d) Cells were transfected with scrambled- or PP2Ac-siRNA and stimulated with IL-1 and/or calyculin A (5 nM) and stimulated as indicated. After 4 h semiquantitative RT-PCR was performed to determine the efficacy of TNF transcription. (e) Cells were treated as in (d). After 16 h TNF secretion was measured in a TNF ELISA. (f) Whole-cell lysates were left untreated or irradiated ex vivo with UVB. In parallel cells were mock treated or irradiated with UVB and PP2Ac was immunoprecipitated. Lysates and precipitates were subjected to an in vitro phosphatase assay using a threonine phosphopeptide as a substrate

Article Snippet: For WB analysis cytosolic and nuclear protein extracts were detected with antibodies against IkB-a, P-IkBa-Ser32/36, P-IKKb-Ser177/181, PP2Ac (L35A5, 5A5, 16A6, no. 2038; Cell Signaling), IKKb (10A9B6; Imgenex) and a-tubulin (DM1A; Neomarkers).

Techniques: Activation Assay, Immunoprecipitation, Transfection, Knockdown, Stable Transfection, Expressing, In Vitro, Kinase Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Irradiation, Ex Vivo, Phosphatase Assay, Phospho-proteomics

Journal: Cell death and differentiation

Article Title: Identification of PP2A as a crucial regulator of the NF-kappaB feedback loop: its inhibition by UVB turns NF-kappaB into a pro-apoptotic factor.

doi: 10.1038/cdd.2008.98

Figure Lengend Snippet: Figure 7 Scheme of interleukin-1 (IL-1) þ ultraviolet-B light (UVB)-induced interruption of the negative feedback loop of nuclear factor-kB (NF-kB). (a) Upon IL-1 stimulation inhibitor of kB kinaseb (IKKb) becomes phosphorylated at Ser 177/181 followed by subsequent phosphorylation of IkBa at Ser 32/36 causing its proteasomal degradation. Released NF-kB translocates into the nucleus to mediate resynthesis of IkBa. In parallel, PP2A dephosphorylates IKKb that allows for stabilization of resynthesized IkBa within the cytoplasm. (b) In case of co-stimulation with UVB, initial IkBa degradation remains unaffected but UVB-mediated inactivation of PP2Ac results in continuous phosphorylation and activation of IKKb. Consequently, persistent phoshorylation and degradation of IkBa disrupts the negative feedback loop of NF-kB. Prolonged NF-kB activation in this specific physiological context triggers enhancement of apoptosis by repression of anti-apoptotic genes and continuous and elevated expression of tumour necrosis factor-a (TNF)

Article Snippet: For WB analysis cytosolic and nuclear protein extracts were detected with antibodies against IkB-a, P-IkBa-Ser32/36, P-IKKb-Ser177/181, PP2Ac (L35A5, 5A5, 16A6, no. 2038; Cell Signaling), IKKb (10A9B6; Imgenex) and a-tubulin (DM1A; Neomarkers).

Techniques: Phospho-proteomics, Activation Assay, Expressing