human map3k7  (Addgene inc)

Journal: Molecular Systems Biology

Article Title: Identification of covalent modifications regulating immune signaling complex composition and phenotype

doi: 10.15252/msb.202010125

Figure Lengend Snippet: Overview of MIP‐APMS experiments performed to study TLR2 activation and drug perturbation of MAPK14 in human immune cells. Lentiviral transduction efficiency of pLOC‐MAPK14 in U937 cells after antibiotic selection (2 d) determined by the GFP fluorescence of IRES‐GFP. Images represent three replicate experiments. Bar represents median % GFP‐positive cells from 3 experiments, error bars s.d. Workflow for IMAP‐MS in primary human macrophages. Interactome of MAPK14 and MAP3K7 in primary human macrophages (measured in quadruplicates) compared against each other. Interactome of MAPK14 in primary human T4 cells (measured in quadruplicates) compared against each other.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1,000): phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, 9211), GAPDH (14C10) rabbit mAb (Cell Signalling, 2118), p38 MAPK (R&D, AF8691), ARHGEF18 (Sigma, HPA042689), MAP3K7 (R&D, MAB5307), FOSB (R&D, AF2214) and anti‐rabbit IgG, HRP‐linked antibody (Cell Signaling, 7074).

Techniques: Activation Assay, Transduction, Selection, Fluorescence

Journal: Molecular Systems Biology

Article Title: Identification of covalent modifications regulating immune signaling complex composition and phenotype

doi: 10.15252/msb.202010125

Figure Lengend Snippet: Evaluation of the effect of bait overexpression on the proteomes of 13 bait cell lines (5,752 total protein IDs). Comparison of the copy numbers of overexpressed bait proteins in the non‐overexpressing cells (U937 WT) and bait‐overexpressing U937 cells. Copy numbers of bait proteins were calculated with the Perseus Plugin based on the LFQ intensity of the respective bait protein. LFQ intensities of endogenous protein and the overexpressed bait proteins are summarized. Evaluation of the effect of bait overexpression on interactor expression, plotted as prey expression difference between the control and bait‐transduced cells. Total protein LFQ intensity in full proteomes across different cell lines. Central band of the boxplot shows the median, boxes represent the IQR, and 3 biological replicates were performed for every condition. Protein expression difference of wildtype U937 versus transduced U937 (MAPK14‐U937; measured in triplicates, bait protein shown in red). 1D Annotation enrichment comparing U937 WT versus transduced U937 (MAPK14‐U937) with significantly changing Corum annotations highlighted in red and infection‐associated pathways highlighted in blue. Luciferase assay of wild‐type U937 and transduced U937 cell lines (MAPK14‐U937, MAP3K7‐U937, TRAF2‐U937) comparing raw intensity counts of non‐activation (orange) versus PAM3CSK4 activation (blue). Bars represent median, error bars represent s.d., and four biological replicates were performed per condition.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1,000): phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, 9211), GAPDH (14C10) rabbit mAb (Cell Signalling, 2118), p38 MAPK (R&D, AF8691), ARHGEF18 (Sigma, HPA042689), MAP3K7 (R&D, MAB5307), FOSB (R&D, AF2214) and anti‐rabbit IgG, HRP‐linked antibody (Cell Signaling, 7074).

Techniques: Over Expression, Expressing, Infection, Luciferase, Activation Assay

Journal: Molecular Systems Biology

Article Title: Identification of covalent modifications regulating immune signaling complex composition and phenotype

doi: 10.15252/msb.202010125

Figure Lengend Snippet: Pie chart showing the proportion of quantified bait/prey proteins (blue) in comparison with background proteins (red) Boxplots with enrichment differences (log 2 ) of bait/prey versus background proteins. Central band of the boxplot shows the median, boxes represent the IQR, and 19 independent replicates (i.e., 19 bait proteins) were used. P ‐values were calculated by t ‐test. Asterisks indicate significant differences **** P ‐value < 0.0001, * P ‐value < 0.05, *** P ‐value < 0.001. Boxplots with P ‐values (−log 10 ) of bait/prey proteins versus background proteins. Central band of the boxplot shows the median, boxes represent the IQR, and 19 independent replicates (i.e., 19 bait proteins) were used. P ‐values were calculated by t ‐test. Asterisks indicate significant differences **** P ‐value < 0.0001, * P ‐value < 0.05, *** P ‐value < 0.001. Impact of interconnected interactors on enrichment differences and P ‐values: Interactor calling was performed for indicated proteins using big (with SYK and MAPK14) and small (without SYK and MAPK14) control groups. Differences of all significant interactors and P ‐values are shown individually for SYK and MAPK14 pull‐downs. Central band of the boxplot shows the median, boxes represent the IQR, and 16 biological replicates were used. Saint probability (probability of the interactor being a true interactor) plotted against the P ‐value from the Student's t ‐test comparing MAPK14 versus control (transduced with His‐Tag only). Significant interactors from t ‐test analysis are colored in turquoise; SAINT interactors (75% probability of interactor being a true interactor) are above the horizontal line of 0.75. Known interactors are colored green. Heatmap with z ‐scored LFQ intensities of significantly interacting proteins for each bait protein (two‐tailed t ‐test, FDR < 0.01, enrichment > 4) clustered by Euclidean distance. Baits are numbered as in Fig . Number of modifications on MAPK14 detected with the indicated open search algorithms in at least one replicate. Open search was performed in MaxQuant (dependent peptide mode), MS Fragger, and Peaks/Taggraph on the drug mode of action dataset of MAPK14 (Fig ). Mass offsets detected at distinct amino acid positions were kept separately. Venn diagram showing the overlap of modifications identified with open search algorithms in at least one replicate. Number of distinct modifications on MAPK14, identified in at least 70% of the replicates with MaxQuant (red), Peaks/Taggraph (violet), MaxQuant & Peaks/Taggraph (green) and with less than 70% valid values (turquoise) in all searches. Comparison of MaxQuant specific searches for MAP3K7, MAPK14, and TRAF2 to dependent peptide analysis (MaxQuant) and PEAKS/Taggraph. Identified and quantified sites are depicted in turquoise, not identified sites in red. Activation of wild‐type and transgenic MAPK14‐U937 cells with PAM3CSK4. Western blotting analysis using antibodies raised against phospho‐MAPK14 and total MAPK14.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1,000): phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, 9211), GAPDH (14C10) rabbit mAb (Cell Signalling, 2118), p38 MAPK (R&D, AF8691), ARHGEF18 (Sigma, HPA042689), MAP3K7 (R&D, MAB5307), FOSB (R&D, AF2214) and anti‐rabbit IgG, HRP‐linked antibody (Cell Signaling, 7074).

Techniques: Transduction, Two Tailed Test, Activation Assay, Transgenic Assay, Western Blot

Journal: Molecular Systems Biology

Article Title: Identification of covalent modifications regulating immune signaling complex composition and phenotype

doi: 10.15252/msb.202010125

Figure Lengend Snippet: Overview of the dynamic and static PPIs and PTMs identified in 17 different cell lines. Global PPI and PTM differences upon PAM3CSK4 activation between prey proteins, and acetylated, methylated, and phosphorylated peptides. Comparison of TRAF2 interactors in native (0.05% NP‐40 in lysis buffer) versus denaturing (6 M GdmCl in lysis buffer). Intensity profile of TRAF2 interactors in different TRAF2 phospho‐variants, normalized to TRAF2 wild‐type intensities. Differential expression of ISG15 in K‐>R TRAF2 mutants versus wild‐type TRAF2 in full proteomes and interactomes. Central band of the boxplot shows the median, boxes represent the IQR, and 2 biological replicates were performed per condition. Analytical size‐exclusion chromatography profile of TRAF2‐ISG15 binding studies. For each analysis, the individual profiles are shown and indicated by color. Coomassie‐stained SDS–PAGE lanes correlate with the approximate retention times in the chromatogram. Western blot analysis of CRISPR‐KO reporter cell lines with antibodies raised against MAP3K7, ARHGEF18, FOSB. Tubulin was used as a loading control. Heatmap of MAP3K7 interactors after treatment with different MAPK14 inhibitors, with significant hits in at least one treatment ( t ‐test, P –value < 0.05) denoted with an asterisk. Treatments were normalized to DMSO control. Heatmap of TRAF2 interactors after treatment with different MAPK14 inhibitors. Treatments were normalized to DMSO control. Intensity profile of MAP3K7 phosphorylation on Ser367, Ser412, and Ser445 after treatment with different MAPK14 inhibitors, normalized to MAP3K7 bait intensity. Central band of the boxplot shows the median, boxes represent the IQR, and four biological replicates were used per condition. P ‐values were calculated by t ‐test. Asterisks indicate significant differences **** P ‐value < 0.0001, * P ‐value < 0.05, *** P ‐value < 0.001.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1,000): phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, 9211), GAPDH (14C10) rabbit mAb (Cell Signalling, 2118), p38 MAPK (R&D, AF8691), ARHGEF18 (Sigma, HPA042689), MAP3K7 (R&D, MAB5307), FOSB (R&D, AF2214) and anti‐rabbit IgG, HRP‐linked antibody (Cell Signaling, 7074).

Techniques: Activation Assay, Methylation, Lysis, Expressing, Size-exclusion Chromatography, Binding Assay, Staining, SDS Page, Western Blot, CRISPR

Journal: Molecular Systems Biology

Article Title: Identification of covalent modifications regulating immune signaling complex composition and phenotype

doi: 10.15252/msb.202010125

Figure Lengend Snippet: Volcano plot representing the interactome of MAP3K7 (measured 16× in biological replicates) compared against all other pull‐downs in the control group. The results of the t ‐tests are represented in volcano plots, which show the protein enrichment versus the significance of the enrichment. Numbers indicate enrichment ranks with the heatmap labels of (C) serving as the legend. Significant interactors of MAP3K7 (two‐tailed t ‐test, FDR < 0.01, enrichment > 4) are colored in blue (novel interactors) and green (known interactors). Interactors of MAP3K7 (blue: novel interactors, green: known interactors) with interconnecting proteins between different baits colored in gray. Heatmap of significant interactors of MAP3K7 upon activation, with significant hits in at least one time point ( t ‐test, P ‐value < 0.05) denoted with an asterisk. Cell activation was performed for 5, 15, and 30 min with the TLR2 ligand PAM3CSK4 (P3C4). Heatmap of MAP3K7 PTMs (phosphorylation) upon activation, with significant hits ( t ‐test, P ‐value < 0.05) in at least one time point denoted with an asterisk. Induction of NFκB determined based on luciferase luminescence in U937 NFκB reporter cells with CRISPR‐Cas9 knockouts of the potential novel interactors of MAP3K7 upon TLR2 activation (each bar represents a mean of four independent measurements; error bars represent the standard deviation; P ‐values were calculated by t ‐test. Asterisks indicate significant differences. *** P ‐value < 0.001, ** P ‐value < 0.01). Data information: Gray boxes indicate missing values. See also Appendix Figs , Tables and .

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1,000): phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, 9211), GAPDH (14C10) rabbit mAb (Cell Signalling, 2118), p38 MAPK (R&D, AF8691), ARHGEF18 (Sigma, HPA042689), MAP3K7 (R&D, MAB5307), FOSB (R&D, AF2214) and anti‐rabbit IgG, HRP‐linked antibody (Cell Signaling, 7074).

Techniques: Protein Enrichment, Two Tailed Test, Activation Assay, Luciferase, CRISPR, Standard Deviation

Journal: Molecular Systems Biology

Article Title: Identification of covalent modifications regulating immune signaling complex composition and phenotype

doi: 10.15252/msb.202010125

Figure Lengend Snippet:

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1,000): phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, 9211), GAPDH (14C10) rabbit mAb (Cell Signalling, 2118), p38 MAPK (R&D, AF8691), ARHGEF18 (Sigma, HPA042689), MAP3K7 (R&D, MAB5307), FOSB (R&D, AF2214) and anti‐rabbit IgG, HRP‐linked antibody (Cell Signaling, 7074).

Techniques: Recombinant, CRISPR, Plasmid Preparation, Clone Assay, Protease Inhibitor, Lysis, Transfection, Reporter Assay, Mutagenesis, Software

Journal: Cell Death & Disease

Article Title: TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers

doi: 10.1038/s41419-024-06654-1

Figure Lengend Snippet: A Schematic of drop-out screen using a custom lentiviral sgRNA Epi-library in glioma stem cells. B Volcano plot representing log2 fold change and −log10 adjusted p -value of each sgRNA abundance comparing final (day 38 or day 35) and reference (day 0) time point in U3013MG or G166 GSC. Positive (essential genes) and negative (non-targeting) control sgRNAs are colored in red and blue, respectively. Dotted lines indicate cut-off used for hit selection. C Venn diagram showing overlap of hits identified in the two GSC screens and common essential genes based on DepMap data (Archilles common essential, version 22Q1). Table shows log2 fold change depletion of best sgRNA of the 19 gene hits in GSCs not essential. Ranking was performed based on the median gene dependency score of CRISPR screens from all DepMap cell lines. D Western blot of U3013MG iCas9 cells showing loss of TAK1 protein 72 h after doxycycline(dox)-induced expression of Cas9. E Cartoon depicting experimental setup of competitive growth assay in iCas9 GSCs. F – I Barplot of competitive growth assay in iCas9 GSCs. Percentage of BFP-positive cells in population was measured by flow cytometry and depicted relative to wells without Cas9 induction (- dox) at each passage. sgNC (non-targeting control sgRNA), sgCTR (targeting control sgRNA cutting outside a coding gene), sgPRMT5/sgMCM2 (essential gene positive control sgRNAs). J Competitive growth assay with complementation by overexpression of wild type TAK1, or catalytically inactive TAK1 K36W mutant. K Cumulative growth assay in ctr (sgCTR) and TAK1 knockout cells (sgMAP3K7) with complementation by overexpression of wild type TAK1, or catalytically inactive TAK1 K36W mutant.

Article Snippet: Gateway compatible entry vector containing human MAP3K7 was obtained from Addgene (23693, pDONR223-MAP3K7).

Techniques: Control, Selection, CRISPR, Western Blot, Expressing, Growth Assay, Flow Cytometry, Positive Control, Over Expression, Mutagenesis, Knock-Out

Journal: Cell Death & Disease

Article Title: TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers

doi: 10.1038/s41419-024-06654-1

Figure Lengend Snippet: A Representative western blots for the indicated proteins of time course experiment of sgMAP3K7_32 expressing U3013MG iCas9 cells upon induction of Cas9 by dox treatment for up to 7 days. B – D Barplot of % Annexin V positive cells ( B and D ) or Caspase-FITC cells ( C ) quantified by flow cytometry 4 days after induction of TAK1 knockout (dox). E Competitive growth assays showing %TAK1 knockout cells over time in the population (measure by BFP abundance) in the presence of a second sgRNA targeting Caspases-1, -8, and -9. sgRNA including gene name is shown on the x-axis. Percentage of BFP-positive cells in population was measured by flow cytometry and depicted relative to wells without Cas9 induction (- dox) at each passage. Dotted line indicates the effect of TAK1 depletion on the population in the presence of a second non-targeting sgRNA (NC). Error bar indicating mean + SD for 3 biological replicates at each time point. F Western blots of different apoptosis markers 4 days after induction of Cas9 expression with doxycycline (dox) in sgCTR, sgMAP3K7_15 and sgMAP3K7_32 expressing cells. G , H Competitive growth assay as ( E ) with second sgRNAs targeting different apoptosis complex members ( G ) or death receptor genes ( H ).

Article Snippet: Gateway compatible entry vector containing human MAP3K7 was obtained from Addgene (23693, pDONR223-MAP3K7).

Techniques: Western Blot, Expressing, Flow Cytometry, Knock-Out, Growth Assay

Journal: Cell Death & Disease

Article Title: TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers

doi: 10.1038/s41419-024-06654-1

Figure Lengend Snippet: A Schematic cartoon of TAK1 depletion using a dTAG-TAK1 degradation system. B Western blots of time course experiment treating dTAG-TAK1 GSCs with 100 nM dTAG V -1 ligand for indicated amount of time. C Barplot of total % Annexin V positive cells quantified by flow cytometry after treatment with 100 nM dTAG V -1 ligand. 2 biological replicates at each time point are shown. Early apoptotic cells are defined as Annexin V + /DAPI- and late apoptotic cells as Annexin V + /DAPI+. D Western blot of apoptosis markers 24 h after treatment with 100 nM dTAG V -1 ligand. E Barplot of total % Annexin V positive cells quantified by flow cytometry after treatment with 100 nM dTAG V -1 ligand for 4 days in dTAG-TAK1 degron cells after knockout of indicated gene. F Barplot of competitive growth assay of dTAG-TAK1 cells expressing BFP and parental GSCs. Fold change of %BFP-positive cells in population after treatment with dTAG V -1 ligand for 7 days is shown relative to DMSO-treated control. G Cumulative growth assay in dTAG- TAK1 degron cells upon knockout of the second indicated gene by CRISPR and treatment with dTAG V -1 ligand. H Barplot depicting fold change of %BFP-positive dTAG-TAK1 cells in population after treatment with dTAG V -1 ligand for 7 days relative to DMSO-treated control and treatment with increasing concentrations of TNF ligand blocking antibody Etanercept. I Western blot of RIPK1 phosphorylation events after treatment with TNFα with or without TAK1 protein depletion. *denotes unspecific band. J Cartoon of molecular response to TAK1 inhibition in TAK1-dependent GSCs.

Article Snippet: Gateway compatible entry vector containing human MAP3K7 was obtained from Addgene (23693, pDONR223-MAP3K7).

Techniques: Western Blot, Flow Cytometry, Knock-Out, Growth Assay, Expressing, Control, CRISPR, Blocking Assay, Phospho-proteomics, Inhibition

Journal: Cell Death & Disease

Article Title: TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers

doi: 10.1038/s41419-024-06654-1

Figure Lengend Snippet: A Heatmap showing % growth inhibition expressed as the relative reduction in cell numbers after 4 days of treatment with HS-276 relative to mean of DMSO-treated controls in 12 GSC lines. GSCs are classified as sensitive (red) or insensitive (blue) based on a significant difference between cell numbers in HS-276 and DMSO treatment conditions. Shown are representative results of 3 biological replicates. Heatmap to the right shows GSVA score of gene expression signature from cell line for mesenchymal, proneural, or classical GBM subtype. B Principal component analysis plot (PCA) of RNAseq data from GSC lines. HS-276 sensitive lines are shown in red, insensitive ones in blue. C Volcano plot of differentially expressed genes between sensitive and insensitive GSC lines ( n = 6 in each group). Significantly higher expressed genes in sensitive GSCs are colored in red, lower expressed genes in blue, and unchanged in gray. Dotted lines indicate cut-off value used to determine deregulated genes (absolute log2 Fold Change of >1 and adjusted p -value of <0.1). D Barplot of the 12 most significantly enriched Hallmark gene signatures in GSCs sensitive to HS-276 treatment (gene set high, n = 513). E Box and wiskers plot of log2 normalized read counts of baseline expression of selected interferon-stimulated genes (ISGs) in sensitive ( n = 6) and insensitive GSCs ( n = 6) measured by RNAseq. Whiskers show minimum and maximum values within group. Boxes indicate median, upper, and lower quartiles. F ELISA of TNFα concentration in 7 days conditions GSCs supernatant (6 biological replicates). nd = not detected. G Barplot of IFNB1, IFNG , and TNF gene expression in GSCs measured by qPCR and normalized to RPLP0 . H Barplot of fold cell expansion of U3013MG treated for 4 days with indicated drugs. I Heatmap of GSVA scores in GCGR-GSC lines. Samples were ranked based on sensitivity signature GSVA score. ID, GCGR patient ID. * indicates GSC lines selected for testing of responsiveness to HS-276 in vitro. J Scatter plot of % growth in 14 GCGR GSCs after 4 days of treatment with HS-276 relative to DMSO against the sensitivity signature GSVA score. Shown is the relative mean of 3 biological replicates (HS-276/DMSO treated). GSCs with significant reduction in cell numbers upon HS-276 treatment are indicated in red. Dotted line indicates separation based on GSVA score into predicted sensitive (positive score) and predicted insensitive (negative score) GSCs and 25% in growth reduction for sensitivity to TAK inhibition by HS-276 treatment. K Scatter plot of MAP3K7 gene knockout effect against sensitivity signature GSVA score from 59 DepMap glioma cell lines.

Article Snippet: Gateway compatible entry vector containing human MAP3K7 was obtained from Addgene (23693, pDONR223-MAP3K7).

Techniques: Inhibition, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, In Vitro, Gene Knockout

Journal: Cell Death & Disease

Article Title: TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers

doi: 10.1038/s41419-024-06654-1

Figure Lengend Snippet: A Histogram of MAP3K7 dependency gene score from 1070 cancer cell lines. Highlighted are lines most sensitive (red) or insensitive (blue) to MAP3K7 depletion. B Heatmap of cell line frequencies in sensitive, insensitive, or other group plotted over different primary disease categories. * indicates significant enrichment in group with p < 0.05 (Fisher’s exact test). C Volcano plot of differentially expressed genes between sensitive ( n = 47) and insensitive ( n = 51) cancer lines. Significantly higher expressed genes in sensitive cell lines are colored in red, lower expressed genes in blue, and unchanged in gray. Dotted lines indicate cut-off value used to determine deregulated genes (absolute log2 Fold Change of >1 and adjusted p -value of <0.1). D Barplot of the 10 most significantly enriched Hallmark gene signatures highly expressed in lines sensitive to MAP3K7 depletion (gene set high, n = 656). E Violin plot of selected genes differentially expressed between cancer cell lines sensitive and insensitive to MAP3K7 depletion. F Heatmap of HS-276 effect on cell growth in 23 cancer cell lines with DepMap gene dependency score and lineage information. G Barplot of fold cell expansion in 8 TAKi sensitive cell lines with Etanercept and Nec-1s cotreatment.

Article Snippet: Gateway compatible entry vector containing human MAP3K7 was obtained from Addgene (23693, pDONR223-MAP3K7).

Techniques:

Journal: Cell Death & Disease

Article Title: FAK mediates LPS-induced inflammatory lung injury through interacting TAK1 and activating TAK1-NFκB pathway

doi: 10.1038/s41419-022-05046-7

Figure Lengend Snippet: A RAW 264.7 (RAW) cells were pretreated with 1 μM PND-1186 for 1 h and then stimulated with 0.5 μg/mL LPS for 30 min. Cell lysates were analyzed for p-TAK1 (Ser412) and p-IKKα/β (Ser176/180) levels. Total TAK1 and IKKβ were used as control. B RAW cells were treated with 0.5 μg/mL LPS for 10 min. Complexes of FAK-TAK1 were detected by immunoprecipitation. C RAW cells were treated with 0.5 μg/mL LPS for the indicated times. Complexes of FAK-TAK1 were detected by immunoprecipitation. D RAW cells were pretreated with 1 μM PND-1186 for 1 h and then exposed to 0.5 μg/mL LPS for 10 min. Complexes of FAK-TAK1 were detected by immunoprecipitation. E RAW cells were pretreated with 2 μM Takinib for 1 h and then exposed to 0.5 μg/mL LPS for 30 min. The phosphorylated ERK, p38 and JNK were examined by western blot assay. Total ERK, p38, and JNK were used as control (TAKi = Takinib). F RAW cells were pretreated with 2 μM Takinib for 1 h and then stimulated with 0.5 μg/mL LPS for 30 min. Cell lysates were analyzed for p-IKKα/β and IκBα levels. Total IKKβ and GAPDH were used as control. G RAW cells were pretreated with 2 μM Takinib for 1 h and then exposed to 0.5 μg/mL LPS for 24 h. IL-6 proteins in the culture medium were measured by ELISA. Data normalized to total proteins and presented as % LPS [Mean ± SEM, 3 independent experiments; *** P < 0.001 compared to LPS]. H RAW cells were pretreated with 2 μM Takinib for 1 h and then exposed to 0.5 μg/mL LPS for 8 h. mRNA levels of IL-6 were measured. Data normalized to β-actin and expressed as % Ctrl [Mean ± SEM, 3 independent experiments; * P < 0.05 compared to LPS]. I RAW cells were transfected with FAK-expressing plasmid. After 24 h, levels of p-FAK and p-TAK1 were detected. Total FAK, TAK1, and GAPDH were used as control. Control cells were transfected with negative control/empty vector (NC = negative control, O/E = overexpression). J 3T3 cells were transfected with FAK-WT-Flag/FAK-Y397F-Flag/TAK1-Myc expressing plasmid, respectively. After 24 h, levels of p-TAK1 were detected using western blot, with total FAK, Flag, Myc, and GAPDH as controls. K , L Cell-free kinase assay showing rhFAK phosphorylates rhTAK1. rhTAK1 was incubated with rhFAK in the presence or absence of ATP (100 μM). The samples were separated by SDS-PAGE and western blotting was used to detect p-TAK1, TAK1, and FAK in panel K . Densitometric quantification of p-TAK1 levels was determined in panel L [Mean ± SEM, 3 independent experiments; ** P < 0.01 compared to rhTAK1]. M , N FAK-expressing RAW cells were treated with 2 μM Takinib for 12 h. IL-6 ( L ) and TNF-α ( M ) proteins in the culture medium were measured by ELISA. Data normalized to total proteins and presented as fold difference compare to NC [TAKi = Takinib; Mean ± SEM, 3 independent experiments; ** P < 0.01 and *** P < 0.001 compared to NC; # P < 0.05 and ## P < 0.01 compared to O/E].

Article Snippet: To overexpress FAK, FAK mutation, and TAK1 in tool cells, three plasmids respectively encoding Flag-labeled wide-type FAK (FAK-WT-Flag), Flag-labeled FAK Y397F mutation (FAK-Y397F-Flag), and Myc-labeled TAK1 (TAK1-Myc) were designed and obtained from OriGene Technologies (Beijing, China).

Techniques: Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Plasmid Preparation, Negative Control, Over Expression, Kinase Assay, Incubation, SDS Page

Journal: Cell Death & Disease

Article Title: FAK mediates LPS-induced inflammatory lung injury through interacting TAK1 and activating TAK1-NFκB pathway

doi: 10.1038/s41419-022-05046-7

Figure Lengend Snippet: A Representative H&E-stained sections of lung tissues harvested from mice following LPS challenge [scale bar = 100 μm]. B Lung injury scores were assessed from histological analyses of mouse lung tissues [Mean ± SEM, n = 8; * P < 0.05 compared to Ctrl; # P < 0.05 compared to LPS]. C Lung wet/dry ratio was determined at 6 h after LPS challenge. [Mean ± SEM, n = 8; * P < 0.05 compared to Ctrl; # P < 0.05 compared to LPS]. D BALF was collected 6 h after LPS challenge and the amounts of proteins were measured. [Mean ± SEM, n = 8; * P < 0.05 compared to Ctrl; # P < 0.05 compared to LPS]. E , F Levels of IL-6 ( E ) and TNF-α ( F ) in BALF samples. [Mean ± SEM, n = 8; ** P < 0.01 and *** P < 0.001 compared to Ctrl; # P < 0.05 compared to LPS]. G , H Levels of IL-6 ( G ) and TNF-α ( H ) in serum samples of mice [Mean ± SEM. N = 8; *** P < 0.001 compared to Ctrl; # P < 0.05 compared to LPS]. I Mice were challenged with intratracheal LPS and treated with PND-1186. Lung tissues were stained for inflammation marker p-p65 (green). Slides were counterstained with DAPI (blue) [scale bar = 100 μm]. J Protein levels of p-FAK, p-TAK1 and IκBα in mouse lung tissues challenged with intratracheal LPS. Total FAK, TAK, and GAPDH were used as control. K Complexes of FAK-TAK1 in mouse lung tissues challenged with intratracheal LPS were detected by immunoprecipitation.

Article Snippet: To overexpress FAK, FAK mutation, and TAK1 in tool cells, three plasmids respectively encoding Flag-labeled wide-type FAK (FAK-WT-Flag), Flag-labeled FAK Y397F mutation (FAK-Y397F-Flag), and Myc-labeled TAK1 (TAK1-Myc) were designed and obtained from OriGene Technologies (Beijing, China).

Techniques: Staining, Marker, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: FAK mediates LPS-induced inflammatory lung injury through interacting TAK1 and activating TAK1-NFκB pathway

doi: 10.1038/s41419-022-05046-7

Figure Lengend Snippet: Upon LPS challenge, activated FAK directly interacts with TAK1 and then phosphorylates TAK1 to activate MAPKs and NFκB signals, which leads to inflammatory cytokine overproduction in both cultured macrophages and lung tissues.

Article Snippet: To overexpress FAK, FAK mutation, and TAK1 in tool cells, three plasmids respectively encoding Flag-labeled wide-type FAK (FAK-WT-Flag), Flag-labeled FAK Y397F mutation (FAK-Y397F-Flag), and Myc-labeled TAK1 (TAK1-Myc) were designed and obtained from OriGene Technologies (Beijing, China).

Techniques: Cell Culture

Journal: Molecular cell

Article Title: Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression

doi: 10.1016/j.molcel.2018.10.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: lentivirus-expressing MAP3K7-shRNAs (human) , Sigma-Aldrich , SHCLNG-NM_145332.

Techniques: Recombinant, Mutagenesis, Kinase Assay, Sequencing, CRAfT Assay, Software