tak1 inhibitor ng  (MedChemExpress)

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: ( A ) Representative image of spleens of 12 week old WT and TAK1[D175A] × Vav-iCre mice (D175A), scale bar = 1 cm. ( B ) Spleen weights of 12 week old WT ( n = 4) and TAK1[D175A] × Vav-iCre mice ( n = 4). Each symbol represents an individual mouse. ( C – E ) As in ( B ) except that splenocyte ( C ), B cell ( D ) and neutrophil ( E ) numbers in the spleen are shown. ( F ) Representative images of axillary (upper panel) and inguinal (lower panel) lymph nodes (LN) of 12 week old WT and TAK1[D175A] × Vav-iCre mice (D175A). ( G ) Total cell numbers in axillary and inguinal LN of 12 week old WT ( n = 4) and TAK1[D175A] × Vav-iCre mice ( n = 4). ( H ) As in ( G ), except B cell numbers in the LN are shown. Statistical significance between the two genotypes was calculated using the unpaired t -test with Welch's correction; * denotes P < 0.05. ( I ) Extracts of BMDM (10 µg protein) from three separate WT mice or three separate TAK1[D175A] × Vav-iCre mice (D175A) (+,+,+) were denatured in SDS, subjected to SDS–PAGE, transferred to PVDF membranes and immunoblotted using the ECL detection system (GE Healthcare) with antibodies recognising TAK1, TAB1, TAB2, TAB3 and GAPDH as a loading control.

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques: SDS Page, Control

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: BMDM from 4 WT (black bars) and 4 TAK1[D175A] (red bars) mice were stimulated for the times indicated with 1 µg/ml Pam 3 CSK 4 ( A – D ), 250 ng/ml R848 ( E – H ) or 100 ng/ml LPS ( I – L ) and the IL-6 ( A , E and I ), IL-10 ( B , F and J ), IL-12p40 ( C , G and K ) and TNF ( D , H and L ) secreted into the culture medium were measured. Bar graphs show mean values and error bars ± standard error of the mean (SEM). Similar results were obtained in eight other independent experiments comparing BMDM from one WT and one TAK1[D175A] mouse.

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques:

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: BMDMs from 4 WT mice (black circles) and 4 TAK1[D175A] mice (red circles) were stimulated for the times indicated with 1 µg/ml Pam3CSK4 ( A – C ), 250 ng/ml R848 ( D – F ) or 100 ng/ml LPS ( G – I ). RNA was extracted from the cells and il6 mRNA ( A , D , G ), il10 mRNA ( B , E , H ) and il12p40 mRNA ( C , F , I ) and levels were measured by quantitative real-time PCR. Results are presented as fold-increase in mRNA relative to the levels in unstimulated samples. Values were normalised to GAPDH mRNA levels. Similar results were obtained in three separate experiments.

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques: Real-time Polymerase Chain Reaction

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: Peritoneal neutrophils from WT (black bars) and TAK1[D175A] (red bars) mice were stimulated for the times indicated with 1 µg/ml Pam3CSK4 ( A – C ), 250 ng/ml R848 ( D – F ) or 100 ng/ml LPS ( G – I ). The IL-6 ( A , D and G ), IL-12p40 ( B , E and H ) and TNF ( C , F and I ) secreted into the culture medium was then measured at the times indicated. The results were obtained with the neutrophils from one WT and one TAK1[D175A] × Vav-iCre mice and the figures show the error bars ± standard error of the mean (SEM) for triplicate determinations. Similar results were obtained in four additional independent experiments each comparing neutrophils from one WT and one TAK1[D175A] mouse.

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques:

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: ( A – C ) BMDM from WT and/or TAK1[D175A] mice were stimulated for the times indicated with 1 µg/ml Pam 3 CSK 4 ( A ), 250 ng/ml R848 ( B ) or 100 ng/ml LPS ( C ). The cell extracts (10 µg protein) were subjected to SDS–PAGE and immunoblotted with antibodies recognising phosphorylated (p) forms of JNK1/2, p38α, IKKα/β, and for all forms of IκBα and p38α MAPK (p38α). Similar results were obtained in 3 separate experiments. ( D – F ) as in A – C , except that BMDM from TAK1[D175A] mice were incubated for 1 h without (−) or with (+) the TAK1 inhibitors NG25 (2 µM) or 5Z-7-oxozeaenol (3 µM).

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques: SDS Page, Incubation

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: Specificity of 5Z-7-oxozeanol and NG25 towards the MAP3K, MAP4K and STE20 families of protein kinases

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques: Activity Assay

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: WT or TAK1 KO THP1 monocytes clone 4 and clone 5 (C4-Mono and C5-Mono) were stimulated for 30 min with 100 ng/ml Pam 3 CSK 4 ( A ) or 100 ng/ml LPS ( B ). ( C – F ) WT and TAK1 KO THP-1 cells were differentiated to a macrophage-like phenotype by stimulation with PMA (see Methods) then stimulated with Pam3CSK4 ( C , D ) or LPS ( E , F ) as in A. ( A – F ) SDS-denatured cell extracts (10 µg protein) were subjected to SDS–PAGE and immunoblotting with antibodies recognising human TAK1, the phosphorylated (p) forms of JNK1/2, p38α MAPK (p38α), IKKα/β and all forms of p38α (p38α). Similar results were obtained in three separate experiments.

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques: SDS Page, Western Blot

Journal: Biochemical Journal

Article Title: TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells

doi: 10.1042/BCJ20220314

Figure Lengend Snippet: WT THP-1 cells (black bars), TAK1 KO THP1 cells clone 4 (white bars-levels are baseline) and TAK1 KO THP1 cells clone 5 (hatched bars) were stimulated for 8 h or 16 h with 100 ng/ml Pam 3 CSK 4 ( A – D ) or 100 ng/ml LPS ( E – H ) and the secretion of IL-6 ( A , E ), IL-8 ( B , F ), macrophage inflammatory protein (MIP)-1β ( C , G ) and TNF ( D , H ) in the culture medium was measured. Experiments were performed with duplicate dishes of cells and the graphs show the average values. Similar results were obtained in three separate experiments.

Article Snippet: The TAK1 inhibitor NG-25 was synthesised by Dr Natalia Shpiro, MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, and 5Z-7-oxozeaenol was purchased from Tocris (#3604).

Techniques: