Journal: International Journal of Molecular Sciences

Article Title: YB-1 Interferes with TNFα–TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling

doi: 10.3390/ijms21197076

Figure Lengend Snippet: Identification of Y-box binding protein-1 interacting proteins. ( A ) Published interaction partners of Y-box binding protein-1 (YB-1) and the corresponding protein sequences that are needed for interaction. Figure modified from Eliseeva et al. 2011 . ( B ) A cDNA library of human mesangial cells was screened with a yeast-2-hybrid screen for interaction partners of YB-1. A full-length YB-1 or a C-terminally truncated construct ΔYB-1 were used as bait. Identified interactions of YB-1 with receptor Notch-3 and SRp30c have been already published [ , ]. ( C ) Clones generated in the yeast-2-hybrid screen that were positive for YB-1 and progranulin (PGRN) interaction were picked and sequenced, allowing us to identify the binding sequence of the protein. ( D ) RAW 264.7 macrophages were pre-incubated with recombinant YB-1 and/or recombinant PGRN, stained for binding of Bt-TNFα (biotinylated tumor necrosis factor α) and analyzed by flow cytometry.

Article Snippet: Cytokine expression into the media secreted by stimulated BMDMs was measured with a BD cytometric bead array (for the cytokines/chemokines CCL5/RANTES, KC, MIG, CCL3/MIP 1α, CCL2/MCP-1, TNFα, and IL 6) as previously described [ ].

Techniques: Binding Assay, Modification, cDNA Library Assay, Construct, Clone Assay, Generated, Sequencing, Incubation, Recombinant, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: YB-1 Interferes with TNFα–TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling

doi: 10.3390/ijms21197076

Figure Lengend Snippet: YB-1 inhibits TNFα binding to TNF receptors (TNFRs). ( A ) RAW 264.7 macrophages were pre-incubated with increasing doses of recombinant Flag-YB-1 and stained for Bt-TNFα-binding. Binding of Bt-TNFα was analyzed with flow cytometry. ( B ) GST-tagged YB-1 deletion mutants were used to identify the minimal sequence required for interaction. Western Blot analysis of purified GST constructs: lane 1—GST-full length YB-1, lane 2—GST-YB-1 Δ1, lane 3—GST-YB-1 Δ3, lane 4—GST-YB-1 Δ4, lane 5—GST-YB-1 Δ5 and lane 6—GST-tag alone. ( C ) RAW 264.7 cells were pre-incubated with GST-YB-1 deletion mutants, stained for the binding of Bt-TNFα, and analyzed with flow cytometry. ( D ) RAW 264.7 macrophages were incubated with 15 µg recombinant Flag-YB-1 and/or 50 ng/mL Bt-TNFα, washed, lysed and bound ligand detected by Western blotting. GAPDH is included as the loading control.

Article Snippet: Cytokine expression into the media secreted by stimulated BMDMs was measured with a BD cytometric bead array (for the cytokines/chemokines CCL5/RANTES, KC, MIG, CCL3/MIP 1α, CCL2/MCP-1, TNFα, and IL 6) as previously described [ ].

Techniques: Binding Assay, Incubation, Recombinant, Staining, Flow Cytometry, Sequencing, Western Blot, Purification, Construct

Journal: International Journal of Molecular Sciences

Article Title: YB-1 Interferes with TNFα–TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling

doi: 10.3390/ijms21197076

Figure Lengend Snippet: TNFα-mediated signaling is influenced by YB-1 and PGRN. ( A ) Bone marrow-derived macrophages (BMDMs) were stimulated with recombinant mTNF (625 pM), PGRN (5 nM), and YB-1 (28 nM) for 10 min. After cell lysis, proteins were blotted and analyzed for the expression of pERK, pp38, NF-κB, pNF-κB, YB-1, and actin as the loading control. Band intensities were quantified and normalized to actin. Values are expressed as relative band intensity (RBI), with the level of activation for TNFα-stimulation set to 100%. ( B ) The pGL4.32 luciferase reporter construct harboring an NF-κB responsive element was introduced into HEKLentiX cells by calcium phosphate precipitation. Recombinant hTNFα was added at the indicated concentrations following the transfer of cells into serum-free starving medium. Stimulation with recombinant proteins was performed as indicated for 5 h, and luciferase activity quantified according the manufacturer’s instructions. * p = 0.05, ** p = 0.01, *** p = 0.001.

Article Snippet: Cytokine expression into the media secreted by stimulated BMDMs was measured with a BD cytometric bead array (for the cytokines/chemokines CCL5/RANTES, KC, MIG, CCL3/MIP 1α, CCL2/MCP-1, TNFα, and IL 6) as previously described [ ].

Techniques: Derivative Assay, Recombinant, Lysis, Expressing, Activation Assay, Luciferase, Construct, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: YB-1 Interferes with TNFα–TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling

doi: 10.3390/ijms21197076

Figure Lengend Snippet: YB-1 and PGRN modulate TNF-induced cell responses. ( A ) BMDMs were stimulated as described above, lysed, and RNA extracted. Gene array analysis of the RNA was used to identify alterations in gene expression between stimulation conditions. Heat map visualization illustrates differentially expressed genes between each of the stimulation conditions compared to the negative control (i.e., unstimulated cells). Additionally, the comparison of TNFα alone with cells stimulated with TNFα, YB-1, and PGRN. Genes regulated >1.5 fold were selected. Nos2 and Ptgs2 are indicated (arrows). ( B ) BMDMs were stimulated with recombinant mTNF (625 pM), PGRN (5 nM), and YB-1 (28 nM) for 24 h. Cells were lysed; RNA was purified and transcribed into cDNA. Taqman gene expression analysis for NosS2, Ptgs2, MCP-1/CCL2, MIP1α/CCL3, RANTES/CCL5, and matrix metallopeptidase 9 (MMP9) were used to validate the results. Gene expression levels are illustrated as relative fold induction, using the gene expression of the negative control as 1. ( C ) BMDMs were stimulated with recombinant mTNF (625 pM), PGRN (5 nM), and YB-1 (28 nM) for 24 h. Cytokines secreted into the media were measured with a cytometric bead array and analyzed by FACS. The cytokines MIG/CXCL9, MIP1α/CCL3, MCP-1/CCL2, RANTES/CCL5, and KC/IL-8 were found to be regulated depending on the stimulus condition. Values are expressed as relative fold induction, using levels of TNFα-induced cytokine secretion as 1.

Article Snippet: Cytokine expression into the media secreted by stimulated BMDMs was measured with a BD cytometric bead array (for the cytokines/chemokines CCL5/RANTES, KC, MIG, CCL3/MIP 1α, CCL2/MCP-1, TNFα, and IL 6) as previously described [ ].

Techniques: Expressing, Negative Control, Recombinant, Purification