Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 1. TAK1 interacts with TGF- receptors. A, endogenous TAK1 inter- acts with TRI and TRII. Whole cell lysates from unstimulated MMC were subjected to immunoprecipitation (IP) with anti-TRI or anti-TRII antibody, as indicated. Normal rabbit IgG was used as a negative control. Immunopre- cipitates and whole cell lysates (CL) not subjected to immunoprecipitation were analyzed by immunoblotting with anti-TAK1 antibody. B and C, coex- pression of TRII with TRI reduces the interaction of TAK1 with TRI. FLAG- TAK1 was coexpressed with V5/His-TRI and HA-TRII, as indicated, in MMC, and cell lysates were subjected to IP with anti-TRI or anti-TRII antibody (B) or with anti-TAK1 antibody (C). Immunoprecipitates and whole cell lysates (CL) not subjected to IP were analyzed by IB with respective anti-FLAG, anti- V5, or anti-HA antibody. D, TGF-1 stimulation triggers the dissociation of the interaction of TAK1 with TRI. MMC grown to subconfluence were rendered quiescent in medium supplemented with 0.5% FBS for 16 h, then stimulated withTGF-1(2ng/ml)fortheindicatedtimes.CelllysatesweresubjectedtoIP with anti-TRI antibody to pull down endogenous TRI, and the interaction of TAK1 with TRI was confirmed by IB with anti-TAK1 antibody. TAK1 and phosphorylated TAK1 were evaluated by IB with anti-TAK1 and anti-p187- TAK1 antibody, respectively.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Immunoprecipitation, Negative Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 2. TAK1 interacts with TRI in a receptor kinase-independent manner. A, schematic diagram of the construction of mutants of TRI. WT, full-length rat TRI; LC, deletion of the kinase domain; SC, deletion of the GS and kinase domain; GS, mutation of five phosphorylation sites in the GS domain; CA, constitutively active mutant. TM and GS indicate transmembrane domain and glycine/serine-rich domain, respectively. V5-His rep- resents V5 and His6 epitope, respectively. B and C, interaction of TAK1 with mutants of TRI. HA-TAK1 was coexpressed in MMC with wild type or respective mutant versions of V5/His-TRI as indicated. Cell lysates were subjected to IP with anti-V5 antibody followed by IB with corresponding anti-HA or anti-His antibody. The expression of transfected genes in whole cell lysates (CL) not subjected to IP was confirmed by IB with anti-V5 and anti-HA antibodies, respectively.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Mutagenesis, Phospho-proteomics, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 3. TAK1 activation is not dependent on receptor kinase activity of TRI. A, coexpression of TRI with TAK1 induces TAK1 phosphorylation at Thr-187 independently of the receptor kinase activity of TRI. HA-TAK1 was coexpressed in MMC with WT or respective mutant versions of V5/His-TRI (CA(constitutivelyactivemutant),GS(phosphorylationsitemutant),LC(dele- tion of the kinase domain), and SC (deletion of the GS and kinase domain)). Cell lysates were subjected to IB with anti-p187-TAK1, anti-HA, and anti-V5 antibodies. B, TGF-1-induced TAK1 phosphorylation does not require recep- tor kinase activity of TRI. MMC grown to subconfluence were rendered qui- escent in medium supplemented with 0.5% FBS for 16 h and pretreated with 10 M TRI inhibitor SB431542 for 1 h before stimulation with TGF-1 (2 ng/ml)fortheindicatedtimes.CelllysatesweresubjectedtoIBwithanti-p187- TAK1 and anti-TAK1 antibodies, respectively. To verify phosphorylation of Smad2 and Smad3, cell lysates were subjected to IB with both anti-p-Smad2 and anti-p-Smad3 antibodies simultaneously. Total Smad2/3 expression was confirmed by IB with anti-Smad2/3 antibody.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Mutagenesis, Expressing

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 4. TRII interferes with the interaction of TRI with TAK1. A, cytoplasmic region of TRII is responsible for its interaction with TAK1. HA-TAK1 was coexpressed in MMC with respective His-tagged WT, kinase-deficient mutant (KD), or cytoplasmic region truncation mutant (C) of TRII. Cell lysates were subjected to IP with anti-His antibody followed by IB with anti-HA and anti-His antibodies. The expression of HA-TAK1 in whole cell lysates (CL) not subjected to IP was confirmed by IB with anti-HA antibody. B, coexpression of TRI and TRII reduces the interaction of TRI with TAK1 and TRI-mediated TAK1 phos- phorylation independent of receptor kinase activity of TRI. FLAG-TAK1 was coexpressed in MMC with respective WT, constitutively active mutant (CA), or phosphorylation site mutant (GS) of TRI with or without coexpression of HA-TRII. Cell lysates were subjected to IP with anti-V5 antibody followed by IB with anti-FLAG, anti-His, and anti-HA antibodies. TAK1 phosphorylation and the expression of the each exogenous gene in whole cell lysates (CL) not subjected to IP were evaluated by IB with anti-p187-TAK1, anti-FLAG, anti-His, and anti-HA antibodies. C, kinase activity of TRII is not required for the reduction of the interaction of TRI with TAK1. HA-TAK1 was coexpressed in MMC with V5/His-tagged WT or mutants of TRI with or without coexpression of kinase-deficient mutant (KD) of TRII. Cell lysates were subjected to IP with anti-V5 antibody followed by IB with anti-HA, anti-TRI, and anti-TRII antibodies. The expres- sion level of exogenous TAK1 in whole cell lysates (CL) was confirmed by IB with anti-TAK1 antibody. D, cytoplasmic region of TRII is required for the reduction of the interaction of TRI with TAK1. FLAG- TAK1, V5/His-TRI-LC, and either HA-tagged WT or cytoplasmic region truncation mutant (C) of HA-TRII were coexpressed in MMC, as indicated. Cell lysates were subjected to IP with anti-V5 antibody followed by IB with anti-FLAG, anti-His, and anti-HA antibodies. The expression of transfected genes in whole cell lysates (CL) not subjected to IP was confirmed by IB with anti-FLAG, anti-His, and anti-HA antibodies. LC, deletion of the kinase domain.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Mutagenesis, Expressing, Activity Assay, Phospho-proteomics, Transfection

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 5. TRI-mediated and TGF-1-induced TAK1 phosphorylation requires its own kinase activity and TAB1. A, TAB1 induces autophospho- rylation of TAK1. FLAG-TAB1 was coexpressed in MMC with WT, kinase-defi- cient mutant (KD; K63W), or phosphorylation site mutant (TA; T187A) of V5/His-TAK1. Cell lysates were subjected to IB with anti-p187-TAK1, anti-p- anti-p184-TAK1, or anti-V5 antibody to evaluate the expression and the phos- phorylationatThr-187andThr-184ofTAK1.B,TRI-mediatedTAK1phospho- rylation is achieved by kinase activity of TAK1. Either WT or kinase-deficient mutant of TAK1 (KD) was coexpressed in MMC with V5/His-TRI and HA-TRII, as indicated. Cell lysates were subjected to IP with anti-V5 antibody followed by IB with anti-FLAG, anti-His, and anti-HA antibodies. Phosphorylation of TAK1 and the expression of each exogenous gene were confirmed by IB of whole cell lysates (CL) with anti-p187-TAK1, anti-FLAG, anti-His, and anti-HA antibodies. C, TAB1 is indispensable for TGF-1-induced TAK1 activation. After transfection of MMC with control siRNA or siRNA specific for TAB1, cells were incubated for 48 h in medium supplemented with 15% FBS and then renderedquiescentinmediumwith0.5%FBSfor16hfollowedbystimulation withTGF-1(2ng/ml)fortheindicatedtimes.CelllysatesweresubjectedtoIB with anti-TAB1, anti-p187-TAK1, and anti-TAK1 antibodies. To verify phospho- rylation of Smad2 and Smad3, cell lysates were subjected to IB with both anti-p-Smad2 and anti-p-Smad3 antibodies (1:1 mixture). Total Smad2/3 expressionwasconfirmedbyIBwithanti-Smad2/3antibody.Equivalentload- ing of each protein sample was verified by IB for -tubulin.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Phospho-proteomics, Activity Assay, Mutagenesis, Expressing, Activation Assay, Transfection, Control, Incubation

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 6. TAB1 is not associated with TRI and TRII. A, TAB1 does not affect the interaction of TAK1 with TRI and TRII. FLAG-TAK1 was coex- pressedinMMCwithMyc-TAB1andV5/His-TRIorHA-TRIIasindicated.Cell lysates were subjected to IP with anti-TAK1 antibody followed by IB with anti-V5, anti-HA, anti-Myc, and anti-FLAG antibodies. The expression level of respective exogenous gene in the whole cell lysates was evaluated by IB with anti-V5, anti-HA, anti-Myc, and anti-FLAG antibodies. B, TAB1 and p187-TAK1 are not associated with TRI. V5/His-TRI was coexpressed in MMC with HA- TAK1 and Myc-TAB1 as indicated. Cell lysates were subjected to IP with anti-V5 antibody followed by IB with anti-p187-TAK1, anti-HA, anti-Myc, and anti-His antibodies. As a control for IP with anti-V5 antibody, cells coexpress- ingV5/His-TAK1andMyc-TAB1wereused(firstlane,indicatedbydashedbox). Normal mouse IgG was used as a negative control. To evaluate the expression level of respective exogenous gene and TAK1 phosphorylation, whole cell lysates (CL) were subjected to IB with anti-p187-TAK1, anti-HA, anti-Myc, and anti-His antibodies. C, TAB1 and p187-TAK1 do not associate with TRII. HA-TRII was coexpressed in MMC with V5/His-TAK1 and Myc-TAB1 as indi- cated. Cell lysates were subjected to IP with anti-HA antibody followed by IB with anti-p187-TAK1, anti-V5, anti-Myc, and anti-HA antibodies. As a control for IP with anti-HA antibody, cells coexpressing HA-TAK1 and Myc-TAB1 were used (first lane, indicated by dashed box). Normal rabbit IgG was used as a negative control. To evaluate the expression level of respective exogenous gene and TAK1 phosphorylation, whole cell lysates (CL) were subjected to IB with anti-p187-TAK1, anti-V5, anti-Myc, and anti-HA antibodies.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Control, Negative Control, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 7. TRAF6 mediates the interaction of TAK1 with TRI and TGF-1-induced TAK1 activation. A, knockdown of TRAF6 by siRNA inhibits TRI-TAK1 interaction. After transfection of MMC with control siRNA () or siRNA specific for TRAF6 (), cells were incubated for 48 h in medium supplemented with 15% FBS. Cell lysates were subjected to IP with anti-TRI antibody followed by IB with anti-TAK1 antibody. IP with normal rabbit IgG was used for the negative control. Knockdown of TRAF6 and equivalent loading of each protein sample were verified by IB for TRAF6 and TAK1, respectively. B, TRAF6 mediates TGF-1-induced TAK1 activa- tion. After transfection of MMC with control siRNA or siRNA specific for TRAF6, cells were incubated for 48 h in medium supplemented with 15% FBS and then rendered quiescent in medium with 0.5% FBS for 16 h followed by stimulation with TGF-1 (2 ng/ml) for the indicated times. Cell lysates were subjected to IP with anti-TRI antibody followed by IB with anti-TAK1 antibody. Knockdown of TRAF6 was verified by IB with anti-TRAF6 antibody. Phosphorylation of TAK1 and MKK3 was evaluated by IB with anti-p187-TAK1, anti-TAK1, anti-p- MKK3, and anti-MKK3 antibodies. Equivalent loading of each protein sample was verified by IB for -tubulin. C, deletion of polyubiquitination site of TAK1 abrogates TAK1 phosphorylation. HA-TRI was coexpressed with either wild type TAK1 (V5/His-TAK1 WT) or polyubiquitination site mutant of TAK1 (V5/His-TAK1-K34R), as indicated. Cell lysates were subjected to IP with anti-HA antibody followed by IB with anti-V5 antibody. Phos- phorylation of TAK1 and the expression of each exogenous gene were confirmed by IB of whole cell lysates (CL) with anti-p187-TAK1, anti-TAK1, and anti-HA antibodies. D, deletion of polyubiquitination site of TAK1 does not affect TAB1-mediated TAK1 phosphorylation. FLAG-TAB1 was coexpressed with either wild type TAK1 (V5/His- TAK1 WT) or polyubiquitination site mutant of TAK1 (V5/His-TAK1-K34R), as indicated. Phosphorylation of TAK1andtheexpressionofexogenousTAK1wereconfirmedbyIBofwholecelllysates(CL)withanti-p187-TAK1 and anti-V5 antibodies.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Knockdown, Transfection, Control, Incubation, Negative Control, Phospho-proteomics, Mutagenesis, Expressing

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 8. The C terminus of TAK1 is required for the interaction of TAK1 with TRI. A, endogenous TAB2 interacts with TAK1 and TRI. Whole cell lysates from unstimulated MMC were subjected to IP with anti-TRI or anti- TAK1 antibody, as indicated. Normal rabbit IgG was used as a negative con- trol. Immunoprecipitates and whole cell lysates (CL) not subjected to immu- noprecipitation were analyzed by immunoblotting (IB) with anti-TAB2 antibody. B and C, deletion of the C terminus of TAK1 abrogates the associa- tion of TAK1 with TRI. HA-TRI was coexpressed in MMC with either FLAG- tagged wild type TAK1 (WT) or C-terminal-truncated mutant TAK1 (C). Cell lysates were subjected to IP with anti-FLAG antibody (B) or anti-HA antibody (C) followed by IB with anti-HA and anti-TAK1 antibodies (B) or anti-FLAG and anti-HA antibodies (C). D, deletion of C terminus of TAK1 abrogates its phospho- rylationmediatedbyTRI.Wholecelllysates(CL)fromBweresubjectedtoIBwith anti-p187-TAK1, anti-FLAG, and anti-HA antibodies. E, deletion of C terminus of TAK1 does not affect TAB1-mediated TAK1 phosphorylation. Myc-TAB1 was coexpressed with FLAG-tagged wild type TAK1 (WT), kinase-deficient mutant TAK1 (KD), or C-terminal-truncated mutant TAK1 (C). Phosphorylation of TAK1 and the expression of each exogenous TAK1 were confirmed by IB of whole cell lysates (CL) with anti-p187-TAK1 and anti-FLAG antibodies.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Mutagenesis, Phospho-proteomics, Expressing

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β (TGF-β1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-β Receptor Kinase Activity in Mesangial Cells

doi: 10.1074/jbc.m109.007146

Figure Lengend Snippet: FIGURE 9. Schematic representation of proposed model for TGF-1-in- duced activation of TAK1 in MMC. Under unstimulated conditions TAK1 interacts with TRI through the complex formation with TAB2 and TRAF6. TGF-1 stimulation-induced formation of hetero-tetrameric complexes of TRI and TRII triggers autopolyubiquitination of TRAF6 that leads to polyu- biquitination of TAK1. TAK1 is released from the receptor complexes and interacts with TAB1, which in turn induces autophosphorylation of TAK1. The activated TAK1 transmits TGF-1 signal to downstream signaling pathways suchastheMKK3-p38cascadeorisrapidlydeactivatedbyphosphatasePP2A. TGF-1-induced TAK1 activation occurs independent of TRI kinase activity, whereas activation of Smad2/3 involves recruitment and phosphorylation by TRI and requires kinase activity of TRI. Activated Smad2/3 is then released from the receptor complex to interact with Smad4 to transmit TGF-1 signals.

Article Snippet: Polyclonal antibodies against MKK3, p-MKK3/6, p-Thr-187-TAK1 (p187-TAK1), p-Thr-184-TAK1, TAB1, and p-Smad3 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Protein-Protein interactions, Activity Assay, Phospho-proteomics

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Fig. 5. Effects of phospholipase C (PLC) (A) and protein kinase C (PKC) (B) antagonists on Wnt agonist 1-induced TAK1 banding 1 (TAB1) expression in beating rat atria. Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983. *p < 0.05 vs. control, #p < 0.05 vs. Wnta1.

Article Snippet: The antibodies used in this study were as follows: anti-β-actin (1:1,000, AP0060; Bioworld Technology, Wuhan, China), polyclonal anti-PKCβ (1:1,000, ENT3756; Elabscience, Wuhan, China), polyclonal anti-PKCγ (1:1,000, E-AB-13012; Elabscience), polyclonal TAB1 antibody WNT/Ca2+ signaling in isolated rat atria Korean J Physiol Pharmacol 2022;26(6):469-478www.kjpp.net 471 (1:1,000, DF7471; Affinity, Changzhou, China), phospho-TAK1 (Thr184/Thr187) antibody (1:1,000, AF4379), polyclonal antiTAK1 (1:500, 12330-2-AP; Proteintech, Wuhan, China), phosphoATF2 (T69/T71) antibody (1:1,000, AP0525; ABclonal, Wuhan, China), polyclonal anti-ATF2 (1:1,000, 11908-1-AP; Proteintech), anti-LEF1 Antibody (1:1,000, DF7570; Affinity), polyclonal antiTCF3 (1:1,000, 14519-1-AP; Proteintech), polyclonal anti-TCF4 (1:2,000, 13838-1-AP; Proteintech), and horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:3,000, AP132P, Nachuan Biotech, Changchun, China).

Techniques: Expressing, Control

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Fig. 6. Effects of phospholipase C (PLC) (A, B) and protein kinase C (PKC) (C, D) antagonists on Wnt agonist 1-induced TAK1 expression in beating rat atria. Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983; p-TAK1, phospho-TAK1; t- TAK1, total-TAK1. *p < 0.05 vs. control, #p < 0.05 vs. Wnta1.

Article Snippet: The antibodies used in this study were as follows: anti-β-actin (1:1,000, AP0060; Bioworld Technology, Wuhan, China), polyclonal anti-PKCβ (1:1,000, ENT3756; Elabscience, Wuhan, China), polyclonal anti-PKCγ (1:1,000, E-AB-13012; Elabscience), polyclonal TAB1 antibody WNT/Ca2+ signaling in isolated rat atria Korean J Physiol Pharmacol 2022;26(6):469-478www.kjpp.net 471 (1:1,000, DF7471; Affinity, Changzhou, China), phospho-TAK1 (Thr184/Thr187) antibody (1:1,000, AF4379), polyclonal antiTAK1 (1:500, 12330-2-AP; Proteintech, Wuhan, China), phosphoATF2 (T69/T71) antibody (1:1,000, AP0525; ABclonal, Wuhan, China), polyclonal anti-ATF2 (1:1,000, 11908-1-AP; Proteintech), anti-LEF1 Antibody (1:1,000, DF7570; Affinity), polyclonal antiTCF3 (1:1,000, 14519-1-AP; Proteintech), polyclonal anti-TCF4 (1:2,000, 13838-1-AP; Proteintech), and horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:3,000, AP132P, Nachuan Biotech, Changchun, China).

Techniques: Expressing, Control

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Fig. 7. Effects of phospholipase C (PLC) (A), protein kinase C (PKC) (B) and TAK1 (C) antagonists on Wnt agonist 1-induced ATF2 expression in beating rat atria. Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983, TI, TAK1 inhibitor, an inhibi- tor of TAK1; p-ATF2, phospho-ATF2; t-ATF2, total-ATF2. *p < 0.05 vs. control, #p < 0.05 vs. Wnta1.

Article Snippet: The antibodies used in this study were as follows: anti-β-actin (1:1,000, AP0060; Bioworld Technology, Wuhan, China), polyclonal anti-PKCβ (1:1,000, ENT3756; Elabscience, Wuhan, China), polyclonal anti-PKCγ (1:1,000, E-AB-13012; Elabscience), polyclonal TAB1 antibody WNT/Ca2+ signaling in isolated rat atria Korean J Physiol Pharmacol 2022;26(6):469-478www.kjpp.net 471 (1:1,000, DF7471; Affinity, Changzhou, China), phospho-TAK1 (Thr184/Thr187) antibody (1:1,000, AF4379), polyclonal antiTAK1 (1:500, 12330-2-AP; Proteintech, Wuhan, China), phosphoATF2 (T69/T71) antibody (1:1,000, AP0525; ABclonal, Wuhan, China), polyclonal anti-ATF2 (1:1,000, 11908-1-AP; Proteintech), anti-LEF1 Antibody (1:1,000, DF7570; Affinity), polyclonal antiTCF3 (1:1,000, 14519-1-AP; Proteintech), polyclonal anti-TCF4 (1:2,000, 13838-1-AP; Proteintech), and horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:3,000, AP132P, Nachuan Biotech, Changchun, China).

Techniques: Expressing, Control

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Fig. 8. Effects of phospholipase C (PLC), protein kinase C (PKC) and TAK1 antagonists on Wnt agonist 1-induced T cell factor (TCF)3 (A–C) and TCF4 (D– F) expression in beating rat atria. Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983, TI, TAK1 inhibitor. *p < 0.05 vs. control, #p < 0.05 vs. Wnta1.

Article Snippet: The antibodies used in this study were as follows: anti-β-actin (1:1,000, AP0060; Bioworld Technology, Wuhan, China), polyclonal anti-PKCβ (1:1,000, ENT3756; Elabscience, Wuhan, China), polyclonal anti-PKCγ (1:1,000, E-AB-13012; Elabscience), polyclonal TAB1 antibody WNT/Ca2+ signaling in isolated rat atria Korean J Physiol Pharmacol 2022;26(6):469-478www.kjpp.net 471 (1:1,000, DF7471; Affinity, Changzhou, China), phospho-TAK1 (Thr184/Thr187) antibody (1:1,000, AF4379), polyclonal antiTAK1 (1:500, 12330-2-AP; Proteintech, Wuhan, China), phosphoATF2 (T69/T71) antibody (1:1,000, AP0525; ABclonal, Wuhan, China), polyclonal anti-ATF2 (1:1,000, 11908-1-AP; Proteintech), anti-LEF1 Antibody (1:1,000, DF7570; Affinity), polyclonal antiTCF3 (1:1,000, 14519-1-AP; Proteintech), polyclonal anti-TCF4 (1:2,000, 13838-1-AP; Proteintech), and horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:3,000, AP132P, Nachuan Biotech, Changchun, China).

Techniques: Expressing, Control

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Fig. 9. Effects of phospholipase C (PLC) (A), protein kinase C (PKC) (B) and TAK1 (C) antagonists on Wnt agonist 1-induced lymphoid enhancer factor 1 (LEF1) expression in beating rat atria. Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983, TI, TAK1 inhibitor. *p < 0.05 vs. control, #p < 0.05 vs. Wnta1.

Article Snippet: The antibodies used in this study were as follows: anti-β-actin (1:1,000, AP0060; Bioworld Technology, Wuhan, China), polyclonal anti-PKCβ (1:1,000, ENT3756; Elabscience, Wuhan, China), polyclonal anti-PKCγ (1:1,000, E-AB-13012; Elabscience), polyclonal TAB1 antibody WNT/Ca2+ signaling in isolated rat atria Korean J Physiol Pharmacol 2022;26(6):469-478www.kjpp.net 471 (1:1,000, DF7471; Affinity, Changzhou, China), phospho-TAK1 (Thr184/Thr187) antibody (1:1,000, AF4379), polyclonal antiTAK1 (1:500, 12330-2-AP; Proteintech, Wuhan, China), phosphoATF2 (T69/T71) antibody (1:1,000, AP0525; ABclonal, Wuhan, China), polyclonal anti-ATF2 (1:1,000, 11908-1-AP; Proteintech), anti-LEF1 Antibody (1:1,000, DF7570; Affinity), polyclonal antiTCF3 (1:1,000, 14519-1-AP; Proteintech), polyclonal anti-TCF4 (1:2,000, 13838-1-AP; Proteintech), and horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:3,000, AP132P, Nachuan Biotech, Changchun, China).

Techniques: Expressing, Control

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Fig. 10. Effects of TAK1 antagonist on Wnt agonist 1-induced atrial natriuretic peptide (ANP) secretion (A) and dynamics (B) in beat- ing rat atria. Data were expressed as means ± SE. n = 6. Cont, control; Wnta1, Wnt agonist 1; TI, TAK1 inhibitor. *p < 0.05 vs. control, #p < 0.05 vs. Wnta1.

Article Snippet: The antibodies used in this study were as follows: anti-β-actin (1:1,000, AP0060; Bioworld Technology, Wuhan, China), polyclonal anti-PKCβ (1:1,000, ENT3756; Elabscience, Wuhan, China), polyclonal anti-PKCγ (1:1,000, E-AB-13012; Elabscience), polyclonal TAB1 antibody WNT/Ca2+ signaling in isolated rat atria Korean J Physiol Pharmacol 2022;26(6):469-478www.kjpp.net 471 (1:1,000, DF7471; Affinity, Changzhou, China), phospho-TAK1 (Thr184/Thr187) antibody (1:1,000, AF4379), polyclonal antiTAK1 (1:500, 12330-2-AP; Proteintech, Wuhan, China), phosphoATF2 (T69/T71) antibody (1:1,000, AP0525; ABclonal, Wuhan, China), polyclonal anti-ATF2 (1:1,000, 11908-1-AP; Proteintech), anti-LEF1 Antibody (1:1,000, DF7570; Affinity), polyclonal antiTCF3 (1:1,000, 14519-1-AP; Proteintech), polyclonal anti-TCF4 (1:2,000, 13838-1-AP; Proteintech), and horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:3,000, AP132P, Nachuan Biotech, Changchun, China).

Techniques: Control

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Fig. 11. Schematic mechanisms by which Wnt agonist 1 (Wnta1) regulates atrial atrial natriuretic peptide (ANP) secretion and me- chanical dynamics. PLC, phospholipase C; PKC, protein kinase C; TAB1, TAK1 banding 1; TCF, T cell factor; LEF1, lymphoid enhancer factor 1.

Article Snippet: The antibodies used in this study were as follows: anti-β-actin (1:1,000, AP0060; Bioworld Technology, Wuhan, China), polyclonal anti-PKCβ (1:1,000, ENT3756; Elabscience, Wuhan, China), polyclonal anti-PKCγ (1:1,000, E-AB-13012; Elabscience), polyclonal TAB1 antibody WNT/Ca2+ signaling in isolated rat atria Korean J Physiol Pharmacol 2022;26(6):469-478www.kjpp.net 471 (1:1,000, DF7471; Affinity, Changzhou, China), phospho-TAK1 (Thr184/Thr187) antibody (1:1,000, AF4379), polyclonal antiTAK1 (1:500, 12330-2-AP; Proteintech, Wuhan, China), phosphoATF2 (T69/T71) antibody (1:1,000, AP0525; ABclonal, Wuhan, China), polyclonal anti-ATF2 (1:1,000, 11908-1-AP; Proteintech), anti-LEF1 Antibody (1:1,000, DF7570; Affinity), polyclonal antiTCF3 (1:1,000, 14519-1-AP; Proteintech), polyclonal anti-TCF4 (1:2,000, 13838-1-AP; Proteintech), and horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:3,000, AP132P, Nachuan Biotech, Changchun, China).

Techniques:

Journal: Heliyon

Article Title: Neuroprotective effects of takinib on an experimental traumatic brain injury rat model via inhibition of transforming growth factor beta-activated kinase 1

doi: 10.1016/j.heliyon.2024.e29484

Figure Lengend Snippet: Impact of Takinib administration on p -TAK1 and TAK1 levels at 24h post-TBI. p -TAK1 and TAK1 were significantly upregulated 24 h following a TBI. Takinib treatment suppressed p -TAK1 and TAK1 levels post-24 h following a TBI, contrary to the control group. No significant variations were observed among the two TBI groups. Quantitative findings utilized the format of mean ± standard deviation (n = 6); ns, no significance, P > 0.05; *, P < 0.05 versus the control group; #, P < 0.05 versus the TBI + vehicle group. Original images of Western blots are presented in Supplementary fig. 1.

Article Snippet: The blot was then subjected to overnight incubation at 4 °C with primary TAK1 and p -TAK1 (Thr187) antibodies (1:200; Santa Cruz Biotech., CA), ZO-1, an inhibitor of NF-κB (IκB-α), cleaved caspase-3 (1:500; Cell Signaling Technology, MA), claudin-5, p65 (1:1,000, Abcam), β-actin, tubulin and histone 3 (1:1000; Proteintech, USA), Bcl-2 and Bax (1:500; Santa Cruz Biotech., CA).

Techniques: Control, Standard Deviation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TAK1-binding Protein 1, TAB1, Mediates Osmotic Stress-induced TAK1 Activation but Is Dispensable for TAK1-mediated Cytokine Signaling

doi: 10.1074/jbc.m807574200

Figure Lengend Snippet: FIGURE 1. TAB1 is dispensable for TNF and IL-1 signaling pathways. Tab1 control (/), Tab1-deficient (/), and Tab1-restored (/ Tab1) MEFs were stimulated with 20 ng/ml TNF or 5 ng/ml IL-1. Activation of JNK and p38 was detected with anti-phospho-JNK and anti-phospho-p38, and the total amounts of JNK and p38 were detected with anti-JNK and anti-p38. Activa- tion of NF-B was monitored by phosphorylation of IB with anti-phospho- IB, degradation of IB with anti-IB, and electrophoretic mobility shift assay (EMSA). TAK1 and TAB1 were detected with anti-TAK1 and anti-TAB1. The asterisks indicate nonspecific bands.

Article Snippet: The following polyclonal antibodies were used: TAK1 and Tab1 described previously (3), Thr(P)187 TAK1 (27) (Cell Signaling), JNK1 (FL), p38 (N-20), I B , p65 (Santa Cruz), phospho-I B, phospho-p38 (Thr180/Tyr182), and AMPK (Cell Signaling).

Techniques: Protein-Protein interactions, Control, Activation Assay, Phospho-proteomics, Electrophoretic Mobility Shift Assay

Journal: Journal of Biological Chemistry

Article Title: TAK1-binding Protein 1, TAB1, Mediates Osmotic Stress-induced TAK1 Activation but Is Dispensable for TAK1-mediated Cytokine Signaling

doi: 10.1074/jbc.m807574200

Figure Lengend Snippet: FIGURE 2. TAB1 is dispensable for TNF- and IL-1- induced activation of TAK1. A, MEFs were stimulated with 5 ng/ml IL-1 (left panels) or 20 ng/ml TNF (right panels). Activation of TAK1 was monitored by immunoblotting with anti-phospho-TAK1. TAK1 and TAB1 were detected with anti-TAK1 and anti- TAB1. The asterisks indicate nonspecific bands. B, MEFs were pretreated with 10 nM calyculin A for 1 h and stimulated with 5 ng/ml IL-1 for 10 min. Activa- tion of TAK1 was analyzed by immunoblots.

Article Snippet: The following polyclonal antibodies were used: TAK1 and Tab1 described previously (3), Thr(P)187 TAK1 (27) (Cell Signaling), JNK1 (FL), p38 (N-20), I B , p65 (Santa Cruz), phospho-I B, phospho-p38 (Thr180/Tyr182), and AMPK (Cell Signaling).

Techniques: Activation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: TAK1-binding Protein 1, TAB1, Mediates Osmotic Stress-induced TAK1 Activation but Is Dispensable for TAK1-mediated Cytokine Signaling

doi: 10.1074/jbc.m807574200

Figure Lengend Snippet: FIGURE 3. TAB1 mediates osmotic stress-induced activation of TAK1 through its C terminus. A, MEFs were stimulated with 0.5 M NaCl. Activation of TAK1 was analyzed by immunoblots. B, schematic diagram of TAB1 and truncated mutants of TAB1. p38- and TAK1-binding sites are shown. C, Tab1 control (/), Tab1-deficient (/) and Tab1-deficient but restored by expressing FLAG-tagged Tab1N (/ FLAG-Tab1N) or FLAG-tagged Tab1C (/ FLAG-Tab1C) MEFs were stimulated with 0.5 M NaCl. Activation of TAK1 was analyzed by immunoblots. The expression of FLAG-TAB1N was detected by the immunoblot with anti-FLAG. The asterisk indicates a nonspecific band. FLAG-TAB1C was too small to detect by immunoblot analysis.

Article Snippet: The following polyclonal antibodies were used: TAK1 and Tab1 described previously (3), Thr(P)187 TAK1 (27) (Cell Signaling), JNK1 (FL), p38 (N-20), I B , p65 (Santa Cruz), phospho-I B, phospho-p38 (Thr180/Tyr182), and AMPK (Cell Signaling).

Techniques: Activation Assay, Western Blot, Binding Assay, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: TAK1-binding Protein 1, TAB1, Mediates Osmotic Stress-induced TAK1 Activation but Is Dispensable for TAK1-mediated Cytokine Signaling

doi: 10.1074/jbc.m807574200

Figure Lengend Snippet: FIGURE 5. TAB1 is essential for concentration-dependent activation of TAK1. A and B, cell lysates from Tab1 control (/) and Tab1-deficient (/) MEFs were incubated and immunoprecipitated at a high or low protein con- centration as described under “Experimental Procedures.” The immunopre- cipitates were incubated in a kinase buffer and analyzed by immunoblots (IB) or by in vitro kinase assay using bacterially expressed MKK6 as an exogenous substrate. C, 293 cells were transfected with expression vectors for HA-TAK1 and TAB1. At 48 h post-transfection, 3 g of cell lysates form HA-TAK1 alone (lane 1) or HA-TAK1 TAB1 (lane 2) transfected cells were loaded onto SDS- PAGE and analyzed by immunoblots. The sizes of the upper bands of exoge- nous and endogenous TAK1 shown in the top panel correspond to the sizes of phosphorylated forms shown in the middle panel.

Article Snippet: The following polyclonal antibodies were used: TAK1 and Tab1 described previously (3), Thr(P)187 TAK1 (27) (Cell Signaling), JNK1 (FL), p38 (N-20), I B , p65 (Santa Cruz), phospho-I B, phospho-p38 (Thr180/Tyr182), and AMPK (Cell Signaling).

Techniques: Concentration Assay, Activation Assay, Control, Incubation, Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Transfection, Expressing, SDS Page