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<p>CYLD is a deubiquitinating enzyme that plays a crucial role in immunity and inflammation as a negative regulator of NF-κB transcription factor and JNK kinase signaling. Defects in either of these pathways contribute to the progression of numerous inflammatory and autoimmune disorders. Therefore, we set out to unravel molecular mechanisms that control CYLD activity in the context of T cell receptor (TCR) signaling. More specifically, we focused on CYLD phosphorylation at Ser418, which can be detected upon immunoblotting of cell extracts with phospho(Ser418)-CYLD specific antibodies. Jurkat T cells stimulated with either anti-CD3/anti-CD28 or PMA/Ionomycin (to mimic TCR signaling) were used as a model system. The role of specific kinases was analyzed using pharmacological as well as genetic approaches. Our initial data indicated that CYLD is directly phosphorylated by the noncanonical IκB kinases (IKKs) IKKε and TANK Binding Kinase 1 (TBK1) at Ser418 upon TCR stimulation. Treatment with MRT67307, a small compound inhibitor for IKKε and TBK1, inhibited TCR-induced CYLD phosphorylation. However, the phospho(Ser418)-CYLD immunoreactive band was still present in CRISPR/Cas9 generated IKKε/TBK1 double knockout cell lines, where it could still be prevented by MRT67307, indicating that the initially observed inhibitory effect of MRT67307 on TCR-induced CYLD phosphorylation is IKKε/TBK1-independent. Most surprisingly, the phospho(Ser418)-CYLD immunoreactive band was still detectable upon immunoblotting of cell extracts obtained from CYLD deficient cells. These data demonstrate the non-specificity of MRT67307 and phospho(Ser418)-CYLD specific antibodies, implying that previously published results based on these tools may also have led to wrong conclusions. We therefore advise to use genetic knockout studies or alternative approaches for a better validation of antibodies and small compound inhibitors. Interestingly, immunoprecipitation with the phospho(Ser418)-CYLD antibody, followed by immunoblotting with anti-CYLD, revealed that CYLD is phosphorylated by IKKε/TBK1 at Ser418 upon T cell stimulation, but that its direct detection with the phospho(Ser418)-CYLD-specific antibody in a western blot is masked by another inducible protein of the same size that is recognized by the same antibody.</p

The HIF-1α IRES efficiently mediates translation during hypoxic conditions

<p><b>Copyright information:</b></p><p>Taken from "The polypyrimidine tract-binding protein stimulates HIF-1α IRES-mediated translation during hypoxia"</p><p>Nucleic Acids Research 2005;33(21):6884-6894.</p><p>Published online 7 Dec 2005</p><p>PMCID:PMC1310900.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Influence of oxygen depletion on HIF-1α and PITSLRE IRES activity. HEK293T cells were transfected with the bicistronic vectors Di-HIF and Di-PITSLRE (), or with a combination of the monocistronic reporter plasmids pSV-Fluc + pSV-HIF-Rluc and pSV-Fluc + pSV-Rluc (). Transfectants were then kept in either 20 or 1% oxygen for 24 h. The bars represent the averages ( = 3) ± SD of Firefly luciferase (Fluc) and luciferase (Rluc) activities in normoxic (open bars) or hypoxic (closed bars) conditions. Bars are representative of three independent transfection experiments

The binding of PTB to the HIF-1α IRES is enhanced in hypoxic conditions

<p><b>Copyright information:</b></p><p>Taken from "The polypyrimidine tract-binding protein stimulates HIF-1α IRES-mediated translation during hypoxia"</p><p>Nucleic Acids Research 2005;33(21):6884-6894.</p><p>Published online 7 Dec 2005</p><p>PMCID:PMC1310900.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Western blot analysis using an anti-HIF-1α antibody (upper panel), and an anti-β-actin antibody as loading control (lower panel) on blots containing total cell extracts derived from HEK293T cells grown in either 20% oxygen (0 h) or 1% oxygen for 8 or 16 h. () Biotinylated HIF-1α IRES RNA-bound to streptavidin beads was incubated with cytoplasmic extracts derived from the HEK293T cells described in (A). After extensive washing, RNA-bound proteins were analyzed by western blotting using an anti-PTB antibody (upper panel). Western blot analysis of the cytoplasmic cell extracts used for RNA affinity chromatography using an anti-PTB antibody (second panel), an anti-β-actin antibody as loading control (third panel) and an anti-poly(ADP-ribose)polymerase antibody, showed equal cytoplasmic PTB expression in all samples. As positive control for poly(ADP-ribose)polymerase expression whole cell extract from normal HEK293T cells (indicated by asterisk) was used

Cell death and IL-1β maturation upon infection of the MF4/4 macrophage cell line with different <i>P. aeruginosa</i> T3SS mutants.

<p>MF4/4 cells were pre-stimulated with 100 ng/ml LPS for 4 h and infected with WT <i>P. aeruginosa</i> or ΔSTY or ΔSTY/ΔPopB mutant strains at a MOI of 100. Non infected cells prestimulated with 100 ng/ml LPS for 4 h were used as a control. <b>A.</b> LDH release in the culture medium was determined by spectrophotometry as described in materials and methods and is expressed as optical density (absorbance) at 490 nm. Results are the mean +/− SD of triplicates and are representative for four independent experiments. <b>B.</b> Caspase-3 activity in cell extracts was measured in a fluorometric assay with Ac-DEVD-AMC and is expressed as change in fluorescence over time (Δf/min). Results are the mean +/− SD of triplicates and are representative of three independent experiments. <b>C.</b> Culture supernatants (SN) were collected and analyzed for the presence of mature IL-1β by immunoprecipitation of IL-1β followed by SDS-PAGE and western blotting. The corresponding total cell lysates were analyzed for the presence of proIL-1β by western blotting. Results are representative of three independent experiments.</p

IL-1β maturation and secretion in lungs of mice infected with different <i>P. aeruginosa</i> T3SS mutants.

<p>C57BL/6 mice were infected intratracheally with 1.1⁶ cfu of WT <i>P. aeruginosa</i> or ΔSTY or ΔSTY/ΔPopB mutants, and after 4.5 h, BALF was isolated and analyzed by SDS-PAGE and western blotting for the presence of mature IL-1β. The corresponding total lung extracts were also analyzed by western blotting for the presence of pro IL-1β. Results are representative of six different mice per experimental condition.</p

Inflammatory cell infiltration and cell death in lungs of mice infected with different <i>P. aeruginosa</i> T3SS mutants.

<p>C57BL/6 mice were infected intratracheally with 1.1⁶ cfu of <i>P. aeruginosa</i> ΔSTY or ΔSTY/ΔPopB strain for 4.5 h. The cells were analyzed by flow cytometry as described in materials and methods. <b>A.</b> The number of macrophages and PMNs in BALF was counted. <b>B.</b> Total numbers of dead macrophages and PMNs were calculated as the sum of check Annexin-V^pos Sytox^neg, Annexin-V^neg Sytox^pos, and Annexin-V^pos Sytox^pos cells. <b>C.</b> The mode of macrophage death was determined by separating Annexin-V^pos Sytox^neg from Annexin-V^neg Sytox^pos cells (n = 6 mice per group; NI were used as a control). The data are expressed as means (+/− SD) and are representative of 3 independent experiments.</p

Survival of mice infected with different <i>P. aeruginosa</i> T3SS mutant bacteria.

<p>A lethal dose of 8.1⁷ cfu of WT <i>P. aeruginosa</i> or ΔSTY or ΔSTY/ΔPopB mutant strains was instillated intratracheally in C57BL/6 mice. (n = 10 for each group, NI  =  non-infected). Mortality was monitored for 24 h. Results are representative of three independent experiments. ***P≤0.001.</p

Motility defect but not lack of flagellin leads to failure in caspase-1 induction.

<p><b>A</b>–<b>E</b>) LPS-primed RAW264.7 macrophages were infected with or without centrifugation with different strains of <i>S</i>. Typhimurium (MOI 150) that have <i>sopE</i> substituted by <i>sopE^m45-tem-1</i>. WT_TEM or T1⁻_TEM either have normal flagella (wildtype flagella), lack flagellin expression (M−F−), or express monomeric flagellin but do not assemble flagella (M−F+). <b>A</b>) SopE^M45-TEM-1 effector translocation into RAW264.7 macrophages was detected by measuring conversion of the TEM-1 beta-lactamase fluorescent substrate CCF2-AM. Values were normalized to the WT_TEM strain. Centrifugation restores effector translocation by WT_TEM^{<b>M−F−</b>} and WT_TEM^{<b>M−F+</b>}. <b>B</b>) Infection was performed with WT_TEM^{<b>M−F−</b>} (left side) or WT_TEM^{<b>M−F+</b>} (right side), respectively, where after cells were washed extensively, fixed and stained with DAPI (blue), phalloidin-TRITC (red), and anti-Salmonella LPS antibody (green) to visualize attachment of bacteria. Cells with attached WT_TEM^{<b>M−F−</b>} or WT_TEM^{<b>M−F+</b>} without (upper panels) or with centrifugation (lower panels), or with WT_TEM, were quantified as shown in C). Scale bar: 50 µm. <b>C</b>) Black circles: not centrifuged; grey circles: with centrifugation. Data shown from two independent experiments performed in duplicate. Black bar: mean of four data points. <b>D</b>) LDH release and <b>E</b>) IL-1 maturation after infection without (black bars) or with centrifugation (grey bars). Experiments were performed in triplicate; mean +/− SD.; n.s.: not significant; *: p-value ≤0.05.</p

SopE and an intact T1 system contribute to flagellin-independent caspase-1 activation.

<p><b>A</b>) SopE is the main effector protein mediating caspase-1 activation in the absence of flagellin. LDH release induced by strains expressing SopE and SopE2 (Δ<i>sipA</i> Δ<i>sopB;</i> SopE/E2, SopE/E2^{<b>M−F−</b>}, and SopE/E2^{<b>M−F+</b>}) is equivalent to LDH release induced by strains additionally lacking SopE2 (Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE2;</i> SopE/E2, SopE/E2^{<b>M−F−</b>}, and SopE/E2^{<b>M−F+</b>}). Note that data shown in A) and C) were obtained from the same experiments. The value for WT in A) was replotted in C) for better comparison. <b>B</b>). The catalytic activity of SopE (infection with SopE^M45 strain) is required for full LDH release. A strain with a catalytically inactive SopE mutant (SopE^M45G168V; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE2</i>) induces the same level of LDH release as a mutant lacking four effector proteins including SopE (Δ4; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE</i> Δ<i>sopE2</i>). <b>C</b>) Mutants lacking four (Δ4; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE</i> Δ<i>sopE2</i>) or eight (Δ8; Δ<i>sipA</i> Δ<i>sopB</i> Δ<i>sopE</i> Δ<i>sopE2</i> Δ<i>sopA</i> Δ<i>sptP</i> Δ<i>spvB</i> Δ<i>spvC</i>) virulence proteins induce LDH release with (Δ4^{<b>M−F+</b>}, Δ8^{<b>M−F+</b>}) or without flagellin (Δ4^{<b>M−F−</b>}, Δ8^{<b>M−F−</b>}), whereas a <i>sipB</i> mutant that lacks the ability for translocon insertion does not. <b>D</b>) IL-1 maturation induced by Δ4, Δ8, Δ4^{<b>M−F+</b>}, Δ8^{<b>M−F+</b>}, Δ4^{<b>M−F−</b>}, and Δ8^{<b>M−F−</b>}. n.d.: not detected. Mean +/− standard deviation of triplicates from at least 2 independent experiments. n.s.: not significant; *: p-value ≤0.05 (paired t-test in panel B; Mann-Whitney U test in panel C). Data shown in D) are representative of 3 independent experiments.</p

Effector- and T1-induced caspase-1 activation in the absence of flagellin is dose-dependent.

<p><b>A</b>) SopE^M45-TEM-1 translocation by strains SopE/E2_TEM (no centrifugation: black circles; centrifugation: open triangles), SopE/E2_TEM^{<b>M−F−</b>} (no centrifugation: open circles; centrifugation: black squares), and T1⁻_TEM (no centrifugation: black triangles; centrifugation: open squares) at different MOI. <b>B</b>) LDH release induced by the same strains as in A) correlates with SopE^M45-TEM-1 translocation in a dose-dependent manner. Data are representative of 3 independent experiments.</p