Journal: Biology of reproduction

Article Title: Tyrosine phosphorylation of caveolin 1 by oxidative stress is reversible and dependent on the c-src tyrosine kinase but not mitogen-activated protein kinase pathways in placental artery endothelial cells.

doi: 10.1095/biolreprod.105.040881

Figure Lengend Snippet: FIG. 7. Activation of the extracellular-signal regulated kinases MAPK1/3 (ERK2/1) is not involved in Tyr14 phosphorylation of caveolin 1 (CAV1) by H2O2 in fetoplacental artery endothelial cells (oFPAEC). Serum- starved oFPAEC were pretreated with or without increasing concentrations of the specific mitogen-activated protein kinase 1 (termed MEK1) inhibitor PD98059 for 60 min, followed by treatment with or without 0.2 mM H2O2 for 20 min. Total cell extracts were prepared and cellular proteins (20 mg/ lane) were analyzed for phosphorylation of MAPK1/3 (a, upper panel) by Western blotting with a phospho-MAPK antibody (1:1000) recognizing activated MAPK1/3 or by Western blotting a specific anti-CAV1 polyclonal antibody (pCAV1 pAb, 1:1000) as described in Figure 2 (b, upper panel). Total MAPK1/3 and CAV1 levels were measured by Western blotting with an anti-panMAPK1/3 antibody (1:2000) and an anti-CAV1 antibody (CAV1 pAb, 1:40 000) for monitoring sample loading (lower panels). Represen- tative blots of phospho-MAPK1/3, Tyr14 phosphorylated CAV1, and total MAPK1/3 and CAV1 of atypical experiment are shown. Data of mean 6 SEM (n 5 3) of phospho-MAPK1/3 and Tyr14 phosphorylated CAV1 are summarized in their corresponding graphs. * P , 0.05 differ significantly from controls.

Article Snippet: Materials Anti-CAV1, anti-mitogen-activated protein kinase (MAPK) 8/9 (also termed Jun-NH2-terminal kinase JNK1/2), anti-MAPK11 (also termed p38mapk), and anti-c-src tyrosine kinase (CSK) rabbit polyclonal antibodies (pAb) and anti-phosphotyrosine (PY99) monoclonal antibody (mAb) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot

Journal: European Journal of Nutrition

Article Title: Different infant formulas can activate toll-like receptor 9 in vitro and inhibit interleukin 6 in human primary intestinal epithelial cells

doi: 10.1007/s00394-024-03507-7

Figure Lengend Snippet: Expression and activity of TLR4 and TLR9 in human primary intestinal epithelial cells. a Western blot analysis of TLR4 and TLR9 in P-IECs, 0 h (basal) and 24 h after stimulation with TLR4 (LPS) or TLR9 agonist (ODN2006). GAPDH was used as reference protein. b Quantitative RT-PCR showing the levels of TLR4 and TLR9 in P-IECs 0 h, 2 h, and 6 h after stimulation with TLR4 (LPS) or TLR9 agonist (ODN2006). GAPDH was used as reference gene. n = 7/group. c, d HEK-Dual cells were stimulated with TLR9 agonist (ODN2006) for 2 h, 6 h, 11 h, and 24 h and the ( c ) optical density (OD 630 ) or ( d ) relative light units (RLUs) were compared with non-treated cells (basal). n = 4/group. e, f HEK-Dual cells were treated with a TLR9 agonist (A, ODN2006) or with inhibitor (INH-18) and agonist (I + A) in two different concentrations, 0.1 µg/ml ( e ) and 1.0 µg/ml ( f ), and were compared to non-treated cells. Luminescence signal was measured with a TECAN luminescence reader. n = 4/group. g MTT assay showing cell viability in different dilutions of the control formula, compared to control (Co) cells incubated with medium. n = 8/group. Data were analyzed by two-way ANOVA ( b–d ) or one-way ANOVA ( g ) followed by Newman-Keuls post hoc test or Student’s t -test ( e, f ) and are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-phospho-MAPK1/2 (Thr202/Tyr204; 1:2000; #9101; Cell Signaling, Ipswich, USA), rabbit monoclonal anti-phospho-IKBA (Ser32; 1:2000; #2859; Cell Signaling), rabbit monoclonal anti-phospho-MAPK14 (Thr180/Tyr182; 1:2000; #4511; Cell Signaling), mouse monoclonal anti-phospho-MAPK8/9 (Thr183/Tyr185; 1:2000; #9255; Cell Signaling), rabbit monoclonal anti-phospho-IRAK4 (Thr345/Ser346; 1:2000; #11,927; Cell Signaling), rabbit monoclonal anti-IL6 (1:2000; #12,153; Cell Signaling), TLR4 Proteintech 1:1000, and TLR4 Novus 1:1000.

Techniques: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR, MTT Assay, Control, Incubation

Journal: European Journal of Nutrition

Article Title: Different infant formulas can activate toll-like receptor 9 in vitro and inhibit interleukin 6 in human primary intestinal epithelial cells

doi: 10.1007/s00394-024-03507-7

Figure Lengend Snippet: IL6 levels were decreased through various infant formulas in human primary intestinal epithelial cells; in contrast IL6 levels were increased due to TLR9 inhibition. The following phosphorylation stages and proteins were investigated by Western blot analysis in human P-IECs: TLR9, TLR4, pMAPK1/2 and MAPK1/2, pMAPK8/9 and MAPK8/9, pp38 and p38, pIRAK4 and IRAK4, IL6, NFKB, pIKB and IKB. HEK-Dual cells were cultivated in medium, PF, CF, and PHF in a 1/25 dilution, with TLR9 and TLR4 inhibitor (0.1 µg/ml) and non-treated HEK-Dual cells served as reference. In addition, the cells were incubated with L. fermentum CECT5716 (LC) (10 10 CFUs) for 30 min. GAPDH was used as reference protein

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-phospho-MAPK1/2 (Thr202/Tyr204; 1:2000; #9101; Cell Signaling, Ipswich, USA), rabbit monoclonal anti-phospho-IKBA (Ser32; 1:2000; #2859; Cell Signaling), rabbit monoclonal anti-phospho-MAPK14 (Thr180/Tyr182; 1:2000; #4511; Cell Signaling), mouse monoclonal anti-phospho-MAPK8/9 (Thr183/Tyr185; 1:2000; #9255; Cell Signaling), rabbit monoclonal anti-phospho-IRAK4 (Thr345/Ser346; 1:2000; #11,927; Cell Signaling), rabbit monoclonal anti-IL6 (1:2000; #12,153; Cell Signaling), TLR4 Proteintech 1:1000, and TLR4 Novus 1:1000.

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

Journal: Biology of reproduction

Article Title: Tyrosine phosphorylation of caveolin 1 by oxidative stress is reversible and dependent on the c-src tyrosine kinase but not mitogen-activated protein kinase pathways in placental artery endothelial cells.

doi: 10.1095/biolreprod.105.040881

Figure Lengend Snippet: FIG. 1. Temporal protein tyrosine phosphorylation in response to H2O2 exposure in fetoplacental artery endothelial cells (oFPAEC)—immunolog- ical identification of tyrosine phosphorylated caveolin 1 (CAV1). a) Serum- starved oFPAEC were treated with 0.2 mM H2O2 for up to 120 min. Total- cell extracts were prepared and cellular proteins (20 mg/lane) were sub- jected to 12% SDS-PAGE fractionation and transferred on Immobilon-P membranes. Total tyrosine phosphorylated proteins were detected by Western blotting (WB) with a specific anti-phosphotyrosine monoclonal antibody (PY99 mAb, 1:1000). b) The oFPAEC were treated with or with- out 0.5 mM H2O2 for 20 or 60 min, total tyrosine phosphorylated proteins were immunoprecipitated (IP). The IP samples were analyzed by Western blotting with a specific anti-CAV1 polyclonal antibody (CAV1 pAb, 1: 40 000).

Article Snippet: Materials Anti-CAV1, anti-mitogen-activated protein kinase (MAPK) 8/9 (also termed Jun-NH2-terminal kinase JNK1/2), anti-MAPK11 (also termed p38mapk), and anti-c-src tyrosine kinase (CSK) rabbit polyclonal antibodies (pAb) and anti-phosphotyrosine (PY99) monoclonal antibody (mAb) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Phospho-proteomics, SDS Page, Fractionation, Western Blot, Immunoprecipitation

Journal: Biology of reproduction

Article Title: Tyrosine phosphorylation of caveolin 1 by oxidative stress is reversible and dependent on the c-src tyrosine kinase but not mitogen-activated protein kinase pathways in placental artery endothelial cells.

doi: 10.1095/biolreprod.105.040881

Figure Lengend Snippet: FIG. 10. Tyr14 phosphorylation of caveolin 1 (CAV1) by H2O2 is medi- ated by a CSK-dependent pathway in fetoplacental artery endothelial cells (oFPAEC). a) Serum-starved oFPAEC were pretreated with or without in- creasing concentrations of a specific CSK inhibitor PP2 for 60 min, fol- lowed by treatment with or without 0.2 mM H2O2 for 20 min. Total cell extracts were prepared and equal amounts of proteins (.200 mg/sample) were used for immunoprecipitation (IP) with a specific anti-CSK poly- clonal antibody (5 mg/sample). Tyrosine phosphorylated CSK in the im- munoprecipitates was detected by immunnoblotting (IB) with a specific anti-phosphotyrosine monoclonal antibody (PY99 mAb, 1:1000, upper panel). The membranes were stripped and reprobed with anti-CSK anti- body (1:500) to monitor sample loading (lower panel). b) Total cellular proteins (20mg/lane) from the same experiment as above were analyzed for CAV1 Tyr14 phosphorylation as described in Figure 2. Representative blots of phospho-CSK, Tyr14 phosphorylated CAV1, and total CSK and CAV1 of atypical experiments are shown. Data of mean 6 SEM (n 5 4) of phospho-CSK and Tyr14 phosphorylated CAV1 are summarized in the lower graphs. Means with different letters differ significantly (P , 0.05).

Article Snippet: Materials Anti-CAV1, anti-mitogen-activated protein kinase (MAPK) 8/9 (also termed Jun-NH2-terminal kinase JNK1/2), anti-MAPK11 (also termed p38mapk), and anti-c-src tyrosine kinase (CSK) rabbit polyclonal antibodies (pAb) and anti-phosphotyrosine (PY99) monoclonal antibody (mAb) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Phospho-proteomics, Immunoprecipitation