anti p stat1 tyr  (Santa Cruz Biotechnology)

Journal: Carcinogenesis

Article Title: Knockdown of ISOC1 inhibits the proliferation and migration and induces the apoptosis of colon cancer cells through the AKT/GSK-3β pathway

doi: 10.1093/carcin/bgz188

Figure Lengend Snippet: The effects of ISOC1 knockdown on the biological function of colon cancer cells are rescued by UCN01. Further verification of the mechanism of ISOC1 in colon cancer cells. The protein level of p-STAT Tyr 701 in colon cancer cell nuclei was increased by ISOC1 knockdown. ( A ) MTS assays. ( B ) Transwell migration assays. ( C ) Cell apoptosis assay. ( D ) Western blot assay. The protein level of p-GSK-3β Ser 9 was upregulated by overexpression of p-AKT Thr 308 . AKT Δ-Thr308 : p-AKT Thr 308 deletion plasmid. AKT P-Thr308 : p-AKT Thr 308 activation plasmid. ( E ) Western blot assay. The protein levels of c-Myc, MMP2 and MMP9 were significantly reduced by ISOC1 knockdown. ( F ) Confocal microscopy and western blot analysis of p-STAT1 Tyr 701 nuclear localization. Green signals represent cells infected with lentiviruses. DNA was visualized with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Western blot analysis showed that the protein levels of p-STAT1 Tyr 701 were increased in the shISOC1 group compared to the shCtrl group. The error bars represent the standard deviation. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for the western blotting included GAPDH (AP0063, Bioworld), ISOC1 (ab118245, Abcam), AKT1/2/3 (ET1609-51, Huabio, China), P-AKT Thr 308 (AF3262, Affinity), P-GSK-3β Ser 9 (AF2016, Affinity), P-STAT1 Tyr 701 (AF6300, Affinity), PARP1 (13371-1-AP, Proteintech), Caspase-3 (9662, CST), Caspase-8 (GB-11594, Servicebio, China), Caspase-9 (GB-11053-1, Servicebio, China), c-Myc (5605s, CST), MMP2 (4022s, CST) and MMP9 (3852s, CST).

Techniques: Knockdown, Migration, Apoptosis Assay, Western Blot, Over Expression, Plasmid Preparation, Activation Assay, Confocal Microscopy, Infection, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Changes in protein expression levels of iNOS and total and phosphorylation levels of STAT1 and STAT3 in LPS-stimulated MG6 cells. (A) Typical Western blots of iNOS, phospho-STAT1 at Tyr-701, total STAT-1, phospho-STAT3 at Tyr-705, and total STAT-3 after challenge with 100 ng/ml LPS. GAPDH served as loading control. (B) Time course of changes in iNOS protein expression after LPS application. (C) Time course of changes in STAT1 phosphorylation after LPS application. (D) Time course of changes in STAT3 phosphorylation after LPS application. The results represent the mean ± SEM for three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. time 0 by t -test.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

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

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Effects of STAT1 siRNA transfection and stattic on protein expression levels of iNOS and total and phosphorylation levels of STAT1 in LPS-stimulated MG6 cells. (A) iNOS, phospho-STAT1 at Tyr-701, and total STAT1 before and 15 h after 100 ng/ml LPS application in the presence of or STAT1 siRNAs (siSTAT1) or the negative control siRNAs (siCTL). (B) Concentration-dependent effect of stattic (0.5–2 μM) on iNOS expression before and 15 h after 100 ng/ml LPS application. (C) Effects of siSTAT1 alone, stattic (2 μM) alone, and combination of siSTAT1 and stattic (1 μM) on iNOS, phosphorylated STAT1, and total STAT1 before and 15 h after 100 ng/ml LPS application. (D) Effect of STAT3 siRNAs (siSTAT3) on iNOS and STAT3 expression before and 15 h after 100 ng/ml LPS application. GAPDH served as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

Techniques: Transfection, Expressing, Phospho-proteomics, Negative Control, Concentration Assay, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Effect of GRK2 siRNA transfection on total and phosphorylation levels of STAT1 and STAT3 in LPS-stimulated MG6 cells. (A) Typical Western blots of phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT3 before and 6 h after 100 ng/ml LPS application in the presence of GRK2 siRNAs (siGRK2) or the negative control siRNAs (siCTL). Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. (B) Phosphorylated levels of STAT1 and STAT3 6 h after 100 ng/ml LPS application when siCTL or siGRK2 was transfected. (C) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then phospho-STAT1 and phospho-STAT3 6 h after 100 ng/ml LPS were detected by Western blot analysis. GAPDH and lamin B served as a cytoplasmic and a nuclear marker, respectively. Shown are representative Western blots from three independent experiments in which the same results were obtained. (D) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 100 ng/ml LPS application. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for three independent experiments. ∗ P < 0.05 by t -test.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

Techniques: Transfection, Phospho-proteomics, Western Blot, Negative Control, Expressing, Control, Isolation, Marker

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Effect of GRK2 siRNA transfection on paclitaxel-induced TLR4 signaling for iNOS expression in MG6 cells. (A) Phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT-3 before and 6 h after 10 μM paclitaxel (PTX) application in the presence of GRK2 siRNAs (siGRK2) or the negative control siRNAs (siCTL). Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (B) Effect of siGRK2 transfection on iNOS protein expression 12 h after 5 μM paclitaxel application. The results represent the mean ± SEM for three independent experiments. ∗∗ P < 0.01 by t -test. In the upper side, typical Western blots are shown. GAPDH served as loading control.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

Techniques: Transfection, Expressing, Negative Control, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Effect of GRK2 siRNA transfection on TLR3-mediated signaling for iNOS expression in MG6 cells. (A) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then the time course of changes in IRF1 levels in each fraction after 50 μg/ml poly(I:C) application was tracked by Western blot analysis. (B) Effect of transfection of GRK2 siRNAs (siGRK2) on nuclear translocation of IRF1 60 min after challenge with 50 μg/ml poly(I:C) was compared with that when the negative control siRNAs (siCTL) was transfected. GAPDH and lamin B served as a cytoplasmic and nuclear marker, respectively. (C) Effect of siGRK2 transfection on IFN-β mRNA expression levels 3 h after 50 μg/ml poly(I:C) application. (D) Effect of siGRK2 transfection on phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT-3 4 h after 50 μg/ml poly(I:C) application. GAPDH served as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (E) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 50 μg/ml poly(I:C) application. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for three independent experiments. ∗ P < 0.05 by t -test.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

Techniques: Transfection, Expressing, Isolation, Western Blot, Translocation Assay, Negative Control, Marker, Control

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Effect of GRK2 siRNA transfection on phosphorylation levels of STAT1 and STAT3 and mRNA levels of iNOS in MG6 cells supplemented with exogenous IFN-β. (A) Time course of changes in phospho-STAT1 at Tyr-701, phospho-STAT3 at Tyr-705, and total STAT3 after supplementation with 10 ng/ml IFN-β. Transfection of siGRK2, but not of siCTL, effectively decreased GRK2 protein expression, and GAPDH was used as loading control. Shown are representative Western blots from three independent experiments in which the same results were obtained. (B) Effect of siGRK2 transfection on expression of iNOS mRNA 12 h after 10 ng/ml IFN-β supplementation in the presence of 10 ng/ml LPS. The mRNA levels were expressed as a fold increase above control normalized GAPDH. The results represent the mean ± SEM for four independent experiments. N.D., not detected. ∗ P < 0.05, ∗∗ P < 0.01 by t- test.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

Techniques: Transfection, Phospho-proteomics, Expressing, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Effect of GRK2 inhibitor treatment on iNOS expression, STAT1/3 activation, and IRF1 activation in microglial cells. GRK2 inhibitor at a concentration of 10 μM was added at 30 min before challenge with 100 ng/ml LPS or 10 ng/ml IFN-β. (A) Expression of iNOS protein before and 15 h after LPS application. (B) STAT1 and STAT3 phosphorylation before and 6 h after LPS application. (C) STAT1 and STAT3 phosphorylation before and 15–30 min after IFN-β supplementation. (D) Expression of IRF1 expression before and 1 h after LPS application. (E) Cytoplasmic (C) and nuclear (N) fractions were isolated, and then changes in IRF1 levels in each fraction before and 1 h after LPS application was tracked by Western blot analysis. GAPDH served as loading control and lamin B was used as a nuclear marker. Shown are representative Western blots from three independent experiments in which the same results were obtained.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

Techniques: Expressing, Activation Assay, Concentration Assay, Phospho-proteomics, Isolation, Western Blot, Control, Marker

Journal: Frontiers in Pharmacology

Article Title: Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells

doi: 10.3389/fphar.2019.00059

Figure Lengend Snippet: Schematic diagram of the regulatory role of GRK2 in TLRs-mediated signaling pathways for iNOS expression in microglial cells. GRK2 positively regulates not only the expression/activation of IRF1 but the activation of STAT1/3, leading to the induction of iNOS transcription. See text for details.

Article Snippet: The membrane was blocked for 60 min at room temperature in 1% bovine serum albumin in Tris-buffered saline containing Tween 20, followed by overnight incubation with primary antibody, anti-iNOS rabbit polyclonal antibody (1:1,000; Cell Signaling, Danvers, MA, United States), anti-STAT1 mouse monoclonal antibody (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-phospho-STAT1 (Tyr-701) mouse monoclonal antibody (1:300; Santa Cruz Biotechnology), anti-GRK2 rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology), anti-STAT3 mouse monoclonal antibody (1:1000; Cell Signaling), anti-phospho-STAT3 (Tyr-705) rabbit monoclonal antibody (1:1000; Cell Signaling), anti-IRF1 rabbit monoclonal antibody (1:1000; Cell Signaling), anti-lamin B1 rabbit polyclonal antibody (1:3000; Proteintech, Rosemont, IL, United States), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10000; Wako Pure Chemical, Osaka, Japan), at 4°C.

Techniques: Protein-Protein interactions, Expressing, Activation Assay

Journal: Molecular Metabolism

Article Title: Regulation of glucose uptake and inflammation markers by FOXO1 and FOXO3 in skeletal muscle

doi: 10.1016/j.molmet.2018.09.011

Figure Lengend Snippet: Effects of FOXOdn overexpression on inflammatory signaling pathways in tibialis anterior muscle . (A) Quantification of STAT1 protein content and phosphorylation after FOXO1dn or (B) FOXO3dn electroporation in tibialis anterior muscle. (C) Representative western blots of STAT1 protein content and phosphorylation after FOXO1dn or FOXO3dn overexpression. (D) Gene expression analysis of chemokine genes after FOXO1dn or FOXO3dn overexpression. (E) Gene expression of key immune cell markers after FOXO1dn or FOXO3dn overexpression. Data are mean ± SEM. n = 11 mice. * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies used for immunoblot analysis were purchased from Cell Signaling Technology (Beverly, MA): Akt (#9272), p-Akt Thr 308 (#4056), p-Akt Ser 473 (#9271), GSK3β (#9315), p-GSK3β Ser 9 (#9323), GS (#3839), p-GS Ser 641 (#3891), mTOR (#2983), p-mTOR Ser 2448 (#5536), 4EBP1 (#9644), p-4EBP1 Thr 37/46 (#2855), p-p70S6K Thr 389 and Thr 421 /Ser 424 (#2708), p70S6K (#9205), p-STAT1 Tyr 701 (#9171), STAT1 (#9172).

Techniques: Over Expression, Electroporation, Western Blot, Expressing