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: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Toll-Like Receptor 9–Dependent AMPK α Activation Occurs via TAK1 and Contributes to RhoA/ROCK Signaling and Actin Polymerization in Vascular Smooth Muscle Cells

doi: 10.1124/jpet.117.245746

Figure Lengend Snippet: Proteins probed for in Western blots This table contains a list of the specific antibodies used in the current article, including dilutions, secondary antibody host species, and the company from which the antibody was purchased.

Article Snippet: Phosphorylated TAK1 Thr184/187 , 1:1000 , Rabbit , Thermo Fisher Scientific/MA5-15073.

Techniques: Western Blot, Transduction

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Toll-Like Receptor 9–Dependent AMPK α Activation Occurs via TAK1 and Contributes to RhoA/ROCK Signaling and Actin Polymerization in Vascular Smooth Muscle Cells

doi: 10.1124/jpet.117.245746

Figure Lengend Snippet: TLR9 agonist ODN2395-dependent phosphorylation of AMPKα is prevented by inhibiting TAK1 in VSMCs. Protein expression analysis was performed for phospho (p)-AMPKαThr172 normalized to total AMPKα after incubation in either vehicle (Veh) or ODN2395 (2 μmol/l), with and without (57)-7-oxozeaenol (100 nmol/l) (A); phospho (p)-TAK1Thr184/187 normalized to total TAK1 after incubation in either vehicle or ODN2395, with and without ODN2088 (20 μmol/l) (B); phospho-AMPKαThr172 normalized to total AMPKα after incubation in either vehicle or ODN2395, with and without bafilomycin A1 (10 nmol/l) (C); and phospho-LKB1Ser428 normalized to total LKB1 after incubation in either vehicle or ODN2395, with and without (57)-7-oxozeaenol (D). Above, representative images of immunoblots; below, densitometric analysis. n = 5–8. One-way ANOVA, *P < 0.05 vs. vehicle.

Article Snippet: Phosphorylated TAK1 Thr184/187 , 1:1000 , Rabbit , Thermo Fisher Scientific/MA5-15073.

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

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Toll-Like Receptor 9–Dependent AMPK α Activation Occurs via TAK1 and Contributes to RhoA/ROCK Signaling and Actin Polymerization in Vascular Smooth Muscle Cells

doi: 10.1124/jpet.117.245746

Figure Lengend Snippet: Schematic summarizing TLR9-AMPKα signaling in VSMCs. TLR9 activation in VSMCs leads to the phosphorylation of AMPKα via TAK1, and activated AMPKα can subsequently trigger RhoA/ROCK, mediating the accumulation and disorganization of F actin. IRAK, interleukin-1 receptor–associated kinase.

Article Snippet: Phosphorylated TAK1 Thr184/187 , 1:1000 , Rabbit , Thermo Fisher Scientific/MA5-15073.

Techniques: Activation Assay, Phospho-proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: HSP70 interacts with TRAF2 and differentially regulates TNFα signalling in human colon cancer cells

doi: 10.1111/j.1582-4934.2009.00716.x

Figure Lengend Snippet: HSP70i over-expression promotes the distribution of TRAF2 in detergent-soluble fractions and decreases the recruitment of RIP1, IKK complex and TAK1 in lipid rafts. (A) HSP70 (inducible [HSP70i] and cognate [HSC70] HSP70) as well as TNFR1, TRAF2, RIP1, TAK1 and IKK complex were recruited into lipid rafts of wild-type HT29 cells 5 min. after TNFα (20 ng/ml) treatments. Lipid rafts were isolated by sucrose density gradient centrifugation. The indicated components in each fraction (1–12) were examined by Western blots (equal volume of fractions was loaded). The raft-localized (Raft) caveolin-1 and the non–raft-localized (Soluble) Rab5 and transferrin receptor (TfR) were examined to display the efficiency of lipid raft isolation. (B) Western blot assay of the dynamic alterations of the indicated components within lipid rafts (combination of fractions 2–4) after TNFα (20 ng/ml) treatments of HT29 cells. (C) Mock (pcDNA3.1-Flag)- or HSP70i-Flag-transfected HT29 cells were treated with 20 ng/ml TNFα for 30 min., and then the lipid rafts (fractions 2–4 combination) and soluble fractions (fractions 10–12 combination) were isolated. The indicated components in equal volumes were detected by Western blots.

Article Snippet: The antibodies against ASK1, Bcl-Xl, caspase 3, caspase 8, caveolin-1, cIAP1, FADD, HA tag, Myc tag, RIP1, TNFR1, TRADD and TRAF2, the antibodies against IκBα, IKKα, IKKβ, JNK1/2, NEMO, p38, p65 sub-unit of NFκB (RelA) and TAK1, and the antibodies against phosphorylated ASK1 (Thr845), MKK4 (Thr261), p65/RelA (Ser536) and TAK1 (Thr184/187) were from Cell Signaling Technology (Beverly, MA).

Techniques: Over Expression, Isolation, Gradient Centrifugation, Western Blot, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: HSP70 interacts with TRAF2 and differentially regulates TNFα signalling in human colon cancer cells

doi: 10.1111/j.1582-4934.2009.00716.x

Figure Lengend Snippet: HSP70i over-expression promotes the activation of ASK1/TAK1, the oligomerization of TRAF2, the association of ASK1/TAK1 with TRAF2 in soluble fractions in response to TNFα. (A, B) HSP70i over-expression promotes TNFα-induced phosphorylation and activation of ASK1 and TAK1. Mock (pcDNA3.1-Flag)- or HSP70i-Flag-transfected HT29 cells were treated with 20 ng/ml TNFα for 30 min. Then the phosphorylated ASK1 (p-ASK1) and TAK1 (p-TAK1) contained in cell lysates (A) were examined by Western blot. In (B), ASK1 and TAK1 contained in cell lysates (100 μg) were immunoprecipitated (IP) with ASK1 and TAK1 Ab as indicated or IgG (as control). The kinase activity of ASK1 and TAK1 was measured by in vitro kinase assays in the presence of recombinant MKK4 as substrate. *, the relative signal intensity of p-ASK1 to total ASK1 in HSP70i-transfected cells 30 min. after TNFα treatment (20 ng/ml) was about 2.64 folds to that in mock-transfected cells; **, the relative signal intensity of p-TAK1 to total TAK1 in HSP70i-transfected cells 30 min. after TNFα treatment (20 ng/ml) was about 2.18 folds to that in mock-transfected cells; ▾, the relative signal intensity of p-MKK4 to input ASK1 in HSP70i-transfected cells 30 min. after TNFα treatment (20 ng/ml) was about 2.88 folds to that in mock-transfected cells; ▾▾, the relative signal intensity of p-MKK4 to input TAK1 in HSP70i-transfected cells 30 min. after TNFα treatment (20 ng/ml) was about 2.72 folds to that in mock-transfected cells. (C) HSP70i promotes the oligomerization of TRAF2 in soluble fractions. Mock (pcDNA3.1-Flag)- or HSP70i-Flag-transfected HT29 cells were transiently transfected with Myc- and HA-Tagged TRAF2. Forty-eight hours later, cells were treated with 20 ng/ml TNFα, and the raft fractions (combination of fractions 2–4) and soluble fractions (combination fractions 10–12) were immunoprecipitated (IP) with HA agarose for TRAF2-HA. The IgG agarose was used as control. The associated TRAF2-Myc was immunoblotted by using Myc Ab. The expression of transfected vectors and markers of raft (Caveolin-1)/soluble (Rab5) fractions were examined by Western blot. (D) HSP70i promotes the association of TRAF2 with ASK1 and TAK1. Cells were treated as in (A). Then TRAF2 in cell lysates (100 μg) was immunoprecipitated with TRAF2 Ab or IgG. The associated ASK1 and TAK1 contained in the immunocomplex were examined by immunoblotting (IB). Otherwise, the immunocomplex was subjected to in vitro kinase assay in the presence of recombinant MKK4. *, the relative signal intensity of TRAF2-associated ASK1 to input TRAF2 in HSP70i-transfected cells 30 min. after TNFα treatment (20 ng/ml) was about 3.24 folds to that in mock-transfected cells; **, the relative signal intensity of TRAF2-associated TAK1 to input TRAF2 in HSP70i-transfected cells 30 min. after TNFα treatment (20 ng/ml) was about 4.03 folds to that in mock-transfected cells; ▾, the relative signal intensity of p-MKK4 to input TRAF2 in HSP70i-transfected cells 30 min. after TNFα treatment (20 ng/ml) was about 5.22 folds to that in mock-transfected cells. (E) HSP70 promotes the association of HSP70 with TRAF2 and the association of TRAF2 with ASK1/TAK1 in soluble fractions. Mock- or HSP70i-Flag-transfected HT29 cells were treated with 20 ng/ml TNFα for 30 min. Then the raft fractions (combination of fractions 2–4) and soluble fractions (combination fractions 10–12) were immunoprecipitated (IP) with HSP70 Ab (for HSP70i and HSC70) and TRAF2 Ab as indicated, or IgG (as control). The associated TRAF2 and ASK1/TAK1 were immunoblotted (IB) as indicated. Markers of raft (Caveolin-1)/soluble (Rab5) fractions were examined by Western blot as loading control.

Article Snippet: The antibodies against ASK1, Bcl-Xl, caspase 3, caspase 8, caveolin-1, cIAP1, FADD, HA tag, Myc tag, RIP1, TNFR1, TRADD and TRAF2, the antibodies against IκBα, IKKα, IKKβ, JNK1/2, NEMO, p38, p65 sub-unit of NFκB (RelA) and TAK1, and the antibodies against phosphorylated ASK1 (Thr845), MKK4 (Thr261), p65/RelA (Ser536) and TAK1 (Thr184/187) were from Cell Signaling Technology (Beverly, MA).

Techniques: Over Expression, Activation Assay, Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation, Control, Activity Assay, In Vitro, Recombinant, Expressing, Kinase Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: HSP70 interacts with TRAF2 and differentially regulates TNFα signalling in human colon cancer cells

doi: 10.1111/j.1582-4934.2009.00716.x

Figure Lengend Snippet: HSP70 silence inhibits TRAF2 ubiquitination, prolongs the presence of TRAF2-RIP1-TAK1-IKK in lipid rafts, promotes RIP1 ubiquitination and NFκB activation and inhibits JNK activation. (A) HT29 cells were transiently silenced with siRNA duplexes specific for HSP70 (HSP70 siRNA, for HSP70i and HSC70) or scrambled control siRNA duplexes (Ctrl siRNA) for 48 hrs. Then HSP70i and HSC70 contained in cell lysates were examined by Western blot. (B) HT29 cells silenced with indicated siRNAs were stimulated with 20 ng/ml TNFα for 5, 30 and 60 min. respectively. Then the raft fractions (combination of fractions 2–4) were examined for the distribution of indicated molecules by Western blot. Raft marker caveolin-1 and soluble fraction marker Rab5 were examined to display the efficiency of raft isolation and the equal loading. (C, D) Silenced HT29 cells were treated with 20 ng/ml TNFα for 30 min. Then TRAF2 and RIP1 in cell lysates (100 μg) were immunoprecipitated (IP) with TRAF2 Ab (C) or RIP1 Ab (D), respectively. Polyubiquitination was examined by immunoblotting (IB) with ubiquitin Ab and HRP-linked pre-adsorbed anti-mouse IgG. Control IgG was also used in the IP processes (data not shown). (E) ELISA assays of phosphorylated IκBα (p-IκBα) in 100 μg lysates derived from silenced HT29 cells that were treated with 20 ng/ml TNFα as indicated. Results were presented as mean OD at 450 nm ± S.D. of triplicate samples. *, P < 0.05; **, P < 0.01 (ANOVA). (F) IKKβ kinase activity assay. IKKβ was immunoprecipitated (IP) with IKKβ Ab or IgG (as control). The in vitro kinase assay was performed by using recombinant IκBα as substrate. *, the relative signal intensity of p-IκBα to input IKKβ in HSP70-silenced cells 30 min. after TNFα treatment (20 ng/ml) was about 1.35 folds to that in Ctrl siRNA-transfected cells; **, the relative signal intensity of p-IκBα to input IKKβ in HSP70-silenced cells 60 min. after TNFα treatment (20 ng/ml) was about 7.48 folds to that in Ctrl siRNA-transfected cells. (G) NFκB gene reporter assay. HT29 cells were transiently co-transfected with pNFκB-Luc, pTA-Luc vectors or siRNA duplexes for 48 hrs. Then cells were treated with 20 ng/ml TNFα for 4h. The luciferase activity was determined by dual-luciferase assays of the lysates. Data are expressed as fold increase relative to untreated mock cells, and presented as mean ± S.D. of triplicate samples. **, P < 0.01 (Student’s t-test). (H), ELISA assays of phosphorylated JNK1/2 (p-JNK1/2) in 100 μg lysates derived from siRNA-silenced cells that were treated with 20 ng/ml TNFα as indicated. Results were presented as mean ± S.D. of triplicate samples. *, P < 0.05; **, P < 0.01 (ANOVA). (I) HT29 cells were transiently silenced with siRNA duplexes specific for HSP70i, HSC70 or corresponding scrambled control siRNA duplexes (Ctrl siRNA) for 48 hrs. Then HSP70i and HSC70 contained in cell lysates were examined by Western blot. (J) Apoptosis assay. HT29 cells were silenced with siRNAs for HSP70i, HSC70 or HSP70 (both HSP70i and HSC70) for 48 hrs, treated with 20 ng/ml TNFα as indicated, stained with annexin V/PI and analyzed by FACS. The apoptotic cells (the annexin V–positive cells) were indicated as the percentages of whole populations. Results were presented as percentage of annexin V + cells to that of whole populations, and expressed as mean ± S.E.M. of three independent experiments. ▴, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (ANOVA).

Article Snippet: The antibodies against ASK1, Bcl-Xl, caspase 3, caspase 8, caveolin-1, cIAP1, FADD, HA tag, Myc tag, RIP1, TNFR1, TRADD and TRAF2, the antibodies against IκBα, IKKα, IKKβ, JNK1/2, NEMO, p38, p65 sub-unit of NFκB (RelA) and TAK1, and the antibodies against phosphorylated ASK1 (Thr845), MKK4 (Thr261), p65/RelA (Ser536) and TAK1 (Thr184/187) were from Cell Signaling Technology (Beverly, MA).

Techniques: Ubiquitin Proteomics, Activation Assay, Control, Western Blot, Marker, Isolation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Derivative Assay, Kinase Assay, In Vitro, Recombinant, Transfection, Reporter Assay, Luciferase, Activity Assay, Apoptosis Assay, Staining