Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 142 Nd cCasp3 D3E9 Fluidigm 3142004A 143 Nd CD19 HIB19 Biolegend 302202 144 Nd pPLCg2 [Y759] K86-689.37 Fluidigm 3144015A 145 Nd CD4 RPA-T4 Fluidigm 3145001B 146 Nd IgD IA6-2 Fluidigm 3146005B 147 Sm CD20 2H7 Fluidigm 3147001B 148 Nd IgA Polyclonal Fluidigm 3148007B 149 Sm CD25 2A3 Fluidigm 3149010B 150 Nd pStat5 [Y694] 47 Fluidigm 3150005A 151 Eu CD123 6H6 Fluidigm 3151001B 153 Eu pStat1 [Y701] 4a Fluidigm 3153005A 154 Sm CD45 HI30 Fluidigm 3154001B 155Gd CD27 L128 Fluidigm 3155001B 156 Gd p38 [T180/Y182] D3F9 Fluidigm 3156002A 157 Gd CD24 ML-5 Biolegend 311102 158 Gd pStat3 [Y705] 4/P-Stat3 Fluidigm 3158005A 159Tb CD11c Bu15 Fluidigm 3159001B 160Gd CD14 M5E2 Fluidigm 3160001B 161 Dy CD141(BDCA-3) AD5-14H12 Miltenyi 130-090-694 162 Dy CD66b 80H3 Fluidigm 3162023B 163 Dy CD56 NCAM16.2 Fluidigm 3163007B 164 Dy IkBa L35A5 Fluidigm 3164004A 165 Ho pCREB [S133] 87G3 Fluidigm 3165009A 166 Er CD16 B73.1 Biolegend 360702 167 Er CD38 HIT2 Fluidigm 3167001B 168 Er CD8 SK1 Fluidigm 3168002B 169 Tm CD45RA HI100 Fluidigm 3169008B 170 Er CD3 UCHT1 Fluidigm 3170001B 171 Yb pERK1/2 [T202/Y204] D13.14.4E Fluidigm 3171010A 172 Yb Anti-Ki-67 B56 Fluidigm 3172024B 174 Yb HLA-DR L243 Fluidigm 3174001B 175Lu CD7 CD7-6B7 Biolegend 343102 176 Yb CD127/IL-7Ra P48-48 Novus Bio MAB306-100 209Bi CD11b/Mac-1 ICRF44 Fluidigm 3209003B Open in a separate window 1 Open channels are not shown but include Pd channels, Cd channel, Pt channels, 89Y,152Sm and 173Yb Whole blood phosphoflow panel1.

Techniques:

Journal: Cell Chemical Biology

Article Title: Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

doi: 10.1016/j.chembiol.2021.05.002

Figure Lengend Snippet: The effect of N-terminal NanoLuciferase, HaloTag, or SNAP-tag additions to IL-23 receptor subunits on IL-23-induced STAT3 phosphorylation STAT3 phosphorylation induced in HEK293T cells transfected with different tagged variants of the IL-23 receptor after a 30-min incubation with increasing concentrations of IL-23. Data are expressed as a percentage of the response obtained with 15 nM IL-23. Values are mean ± SEM from four or three (NL-IL23R and HT-IL12Rβ1) independent experiments.

Article Snippet: AlphaLISA SureFire Ultra p-STAT3 (Tyr705) Assay Kit , Perkin Elmer , Cat# ALSU-PST3.

Techniques: Transfection, Incubation

Journal: Cell Chemical Biology

Article Title: Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

doi: 10.1016/j.chembiol.2021.05.002

Figure Lengend Snippet:

Article Snippet: AlphaLISA SureFire Ultra p-STAT3 (Tyr705) Assay Kit , Perkin Elmer , Cat# ALSU-PST3.

Techniques: Recombinant, Modification, Staining, Purification, Luciferase, Expressing, Plasmid Preparation, Software

Journal: Cancer Management and Research

Article Title: Long noncoding RNA PVT1 promotes hepatoblastoma cell proliferation through activating STAT3

doi: 10.2147/CMAR.S213707

Figure Lengend Snippet: PVT1 activates STAT3 and promotes cell cycle progression. ( A ) Western blotting analysis of p-STAT3 and STAT3 in HepG2 and HuH-6 cells stably expressing Lenti-vec or Lenti-PVT1. ( B ) Western blotting analysis of p-STAT3 and STAT3 in HepG2 and HuH-6 cells stably expressing shCtrl or PVT1 shRNA (shPVT1 #1 and shPVT1 #2). The representative images (top) and quantification of p-STAT3 level relative to STAT3 (bottom) are shown. ( C-D ) qRT-PCR analysis of mRNA levels of cyclin D1, myc, and cyclin B1 in HepG2 and HuH-6 cells shown as in ( A-B ). ( E-F ) The cell cycle distribution of HepG2 and HuH-6 cells shown as in ( A-B ) was evaluated by FACS analysis. Data are mean ± SD (n=3). ** P <0.01; * P <0.05 versus control. Abbreviations: STAT3, signal transducer and activator of transcription 3; FACS, fluorescence activated cell sorter.

Article Snippet: The primary antibodies against p-STAT3 (Tyr705) (mouse monoclonal, NBP2-24463) and STAT3 (mouse monoclonal, MAB1779) were purchased from Novus Biologicals.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Control, Fluorescence

Journal: Cancer Management and Research

Article Title: Long noncoding RNA PVT1 promotes hepatoblastoma cell proliferation through activating STAT3

doi: 10.2147/CMAR.S213707

Figure Lengend Snippet: PVT1 promotes hepatoblastoma cell proliferation through activating STAT3-mediated cell cycle progression. ( A-E ) HepG2 and HuH-6 cells stably expressing Lenti-vec or Lenti-PVT1 were treated with vehicle control or STAT3 inhibitor stattic. ( A ) The levels of p -STAT3 and STAT3 were determined by Western blotting analysis. The representative images (top) and quantification of p -STAT3 level relative to STAT3 (bottom) are shown. ( B ) The mRNA levels of cyclin D1, myc, and cyclin B1 were determined by qRT-PCR analysis. ( C ) The cell cycle distribution was evaluated by FACS analysis. #, P <0.01, Lenti-PVT1 versus Lenti-vec; &, P <0.01, Lenti-PVT1 plus stattic versus Lenti-PVT1 plus vehicle control. ( D-E ) The cell proliferation of HepG2 ( D ) and HuH-6 ( E ) cells was monitored by CCK-8 assay during the course of continuous cell culture. Data are mean ± SD (n=5). ** P <0.01; NS, not significant.

Article Snippet: The primary antibodies against p-STAT3 (Tyr705) (mouse monoclonal, NBP2-24463) and STAT3 (mouse monoclonal, MAB1779) were purchased from Novus Biologicals.

Techniques: Stable Transfection, Expressing, Control, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Cell Culture

Journal: Rheumatology (Sunnyvale, Calif.)

Article Title: Monosodium Urate and Tumor Necrosis Factor-α Increase Apoptosis in Human Chondrocyte Cultures

doi: 10.4172/2161-1149.1000113

Figure Lengend Snippet: Western blotting for p38 kinase, JNK and STAT3. Human OA chondrocytes were grown to hyperdensity as previously described [ , ] and treated with rhTNF-α (10ng/ml) or not (C) for up to 60 min. Cytosolic protein lysate from the 1, 5, 30 and 60 min incubations were electrophoresed as previously described and Western blotting performed . Panels A and B: Western blots were produced as previously described using anti-phospho-p-38 MAPK antibody or an anti-p38 MAPK antibody; ( Panel A ), anti-phospho-SAPK p54 (JNK 2) antibody/anti-phospho-SAPK p46 (JNK 1) antibody or ( Panel B ) anti-SAPK p54 (JNK 2)/anti-SAPK p46 (JNK 1) antibody. Panel C: A Western blot was produced with the specific anti-U-STAT3 antibody or anti-phospho-STAT3 antibody as indicated in Materials and Methods. The bands migrating faster than the phospho-STAT3 standard have retained reactivity with anti-phospho-STAT3, but have not been fully characterized. These bands may represent degradation products of phospho-STAT3. The fastest migrating band reacting with anti-phospho-STAT3 was also present in the control (C) group.

Article Snippet: For immunohistochemical detection of STAT3, an anti-STAT3 monoclonal antibody that detects endogenous human, mouse and rat unphosphorylated STAT3 (U-STAT3) and an affinity-purified rabbit anti-STAT3 antibody that detects STAT3 phosphorylated at Y705 (p-STAT3) were obtained from R and D Systems.

Techniques: Western Blot, Produced, Control

Journal: Rheumatology (Sunnyvale, Calif.)

Article Title: Monosodium Urate and Tumor Necrosis Factor-α Increase Apoptosis in Human Chondrocyte Cultures

doi: 10.4172/2161-1149.1000113

Figure Lengend Snippet: U-STAT3 and p-STAT3 in normal juvenile chondrocyte pellet cultures (N-JHu-C) and pellet cultures initiated from MSCs (BMD-MSC-C).

Article Snippet: For immunohistochemical detection of STAT3, an anti-STAT3 monoclonal antibody that detects endogenous human, mouse and rat unphosphorylated STAT3 (U-STAT3) and an affinity-purified rabbit anti-STAT3 antibody that detects STAT3 phosphorylated at Y705 (p-STAT3) were obtained from R and D Systems.

Techniques:

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV results in activated phosphorylation of STAT3 on Tyr705 and diaphragm contractile dysfunction. Diaphragms from mechanically ventilated (MV) rats and unfed controls were analyzed. A ) Ex vivo force–frequency relationship from mechanically ventilated ( n =13) rats and unfed controls ( n =7). B ) Western blots for total STAT3 and phospho-STAT3 Y705 from a representative set of mechanically ventilated rats and unfed controls (1, 3, 6, and 9 h, n =5–6 rats/group; 18 h, n =6–8). C ) Messenger RNA levels of the STAT3 downstream target genes SOCS3 and Myf5 from mechanically ventilated ( n =9) rats and unfed controls ( n =10). Mechanical ventilation period of 18 h. Results are means ± sem . * P < 0.05; Student's t test.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Ex Vivo, Western Blot

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV increases Ser705 phosphorylation and mitochondrial accumulation of phospho-STAT3 (Ser727 and/or Tyr705/Ser727) within diaphragm muscle. A ) Diaphragms from unfed control rats ( n =8), MV-R548 rats ( n =9), or MV-Veh rats ( n =9) were analyzed by Western blot of total muscle lysate (representative animals depicted in blot) and quantitation. Correlation between increases in phospho-STAT3 S727 and phospho-STAT3 Y705 were determined. B ) Purified mitochondrial fractions (mito) and total lysates (total) were subjected to Western blot analysis for pSTAT3 S727 , pSTAT3 Y705 , total STAT3, GRIM-19, VDAC (mitochondrial marker), LDHA (cytoplasmic marker) and PCNA (nuclear marker). Unfed (U) controls, n = 10; MV-Veh (MV), n = 6; MV-R548, n = 7. Results are means ± sem . Mechanical ventilation period of 18 h. P values calculated by 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Western Blot, Quantitation Assay, Purification, Marker

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: Proposed model for role of JAK signaling in VIDD. JAK signaling is activated by CMV and functions as a critical triggering mechanism upstream of STAT3 phosphorylation (Tyr705 and Ser705), mitochondrial dysfunction, ROS production, atrophy, and muscle weakness. Mechanical ventilation results in the mitochondrial accumulation of phospho-STAT3 (singly phosphorylated on Ser727 or doubly phosphorylated at both Ser727 and Tyr705). Import into mitochondria is facilitated through interaction of phospho-STAT3 with GRIM-19, a component of complex I of the ETC, and may directly affect mitochondrial function and ROS generation. Induction of various myogenic transcription factors and muscle-specific E3 ubiquitin ligases (MURF-1 and atrogin-1) and activation of calpain, caspase 9, and caspase 3 can contribute to muscle atrophy and proteolytic cleavage of myofilament proteins. Mitochondrial dysfunction may also lead to ROS-mediated modification of myofilament proteins in a manner that negatively affects contractile function.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Activation Assay, Modification