rabbit polyclonal antibody against pstat3  (Cell Signaling Technology Inc)

Journal: Cancer gene therapy

Article Title: STAT3 regulates miR93-mediated apoptosis through inhibiting DAPK1 in renal cell carcinoma.

doi: 10.1038/s41417-020-00235-y

Figure Lengend Snippet: Fig. 5 STAT3 inhibited the mRNA and protein level of DAPK1, and DAPK1 deactivated STAT3. a Relative expression of DAPK1 measured in qRT-PCR after two cell lines treated by siRNA. b Relative expression of DAPK1 measured in qRT-PCR when STAT3 inactivation. c The protein expression of DAPK1 among different groups with STAT3 knockdown and inactivation. d pSTAT3 in nuclear proteins extracted were measured by western blot analysis. e Immunofluorescence assay showed that DAPK1 attenuated pSTAT3 entry into nuclear.

Article Snippet: Then, the cells were reacted with rabbit polyclonal antibody against pSTAT3 (1:200, #9145, Cell Signaling Technology, Danvers, MA, USA) in blocking buffer overnight.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot

Journal: Cancer gene therapy

Article Title: STAT3 regulates miR93-mediated apoptosis through inhibiting DAPK1 in renal cell carcinoma.

doi: 10.1038/s41417-020-00235-y

Figure Lengend Snippet: Fig. 6 STAT3 regulates miR93-mediated apoptosis through inhi- biting DAPK1 in Renal cell carcinoma. STAT3 promotes miR-93-3p expression by binding to promoter region, then miR-93-3p suppressed DAPK1. What’s more, DAPK1 mediates the activation of STAT3 pathway through blocking pSTAT3 translation into the nucleus, and STAT3 decreased the expression of DAPK1.

Article Snippet: Then, the cells were reacted with rabbit polyclonal antibody against pSTAT3 (1:200, #9145, Cell Signaling Technology, Danvers, MA, USA) in blocking buffer overnight.

Techniques: Expressing, Binding Assay, Activation Assay, Blocking Assay

Journal: Molecular cancer therapeutics

Article Title: Sphingosine-1-Phosphate Receptor-1 Promotes Environment-Mediated and Acquired Chemoresistance

doi: 10.1158/1535-7163.MCT-17-0379

Figure Lengend Snippet: (A) Western blotting showing expression of indicated proteins in CHLA-255 cells after 24 h stimulation with BM-MSC CM. Data are representative of 3 independent experiments with similar results. Right panel, immunofluorescent images showing S1PR1 expression and nuclei in CHLA-255 cells stimulated for 1.5 h with BM-MSC CM. Shown are representative images of 3 independent experiments. Scale bar, 10 µm. (B) pSTAT3 protein levels in CHLA-255 cells transduced with lentiviral vectors containing nontargeting (NT) shRNA control or two S1PR1 shRNA variants (#1 and #2). Data are representative of 3 independent experiments with similar results. (C) Flow cytometry analysis of apoptosis in CHLA-255 cells expressing the indicated shRNAs after treatment with etoposide (1 µM) for 24 h. (D) Representative Annexin V staining by flow cytometry of CHLA-255 cells expressing NT or S1PR1 shRNA co-cultured with BM-MSC followed by exposure to 1 µM etoposide. Lower panel, the relative apoptotic index of the NB cells calculated as the percentage of the apoptotic cells co-cultured with BM-MSC relative to the percentage of the apoptotic cells without co-culture. In panel C and D, data represent mean ± SEM from 3 independent experiments. (E) Western blotting shows pSTAT3 and STAT3 expression levels in CHLA-255 cells stably expressing NT or S1PR1 shRNA (#2) stimulated with BM-MSC CM for 20 min or overnight (ON). Data are representative of 3 independent experiments with similar results. In all western blots, band densitometry was normalized to GAPDH or b-actin using ImageJ software and the values are indicated below the gel figures.

Article Snippet: Rabbit polyclonal antibodies against pSTAT3 (Y705) (#9131), pJAK2 (#3771), cleaved PARP-1 (#9542), cleaved caspase-3 (9664), Bcl- X L (sc-8392), Bcl-2 (#2876) and survivin (#2808) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Transduction, shRNA, Flow Cytometry, Staining, Cell Culture, Co-Culture Assay, Stable Transfection, Software

Journal: Molecular cancer therapeutics

Article Title: Sphingosine-1-Phosphate Receptor-1 Promotes Environment-Mediated and Acquired Chemoresistance

doi: 10.1158/1535-7163.MCT-17-0379

Figure Lengend Snippet: (A) Western blotting showing S1PR1, pSTAT3 and STAT3 expression levels in control (vector alone) and MSCV-S1pr1-overexpressing CHLA-255 and CHLA-171 cells. Data are representative of 3 independent experiments with similar results. (B) Flow cytometry analysis of control and S1pr1 overexpressing CHLA-255 cells treated with 1 µM etoposide or 0.05 µM topotecan for 24 h, and then stained with Annexin V. (C) Graph showing percentages of Annexin-V-positive apoptotic cells in control and S1pr1 overexpressing NB cells treated with indicated drugs. Data represent mean ± SEM of 3 independent experiments. (D) Western blotting of uncleaved and cleaved PARP-1 and cleaved caspase-3 in control and S1pr1 overexpressing CHLA-255 cells following exposure to the indicated concentrations of etoposide for 24 h.

Article Snippet: Rabbit polyclonal antibodies against pSTAT3 (Y705) (#9131), pJAK2 (#3771), cleaved PARP-1 (#9542), cleaved caspase-3 (9664), Bcl- X L (sc-8392), Bcl-2 (#2876) and survivin (#2808) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Staining

Journal: Molecular cancer therapeutics

Article Title: Sphingosine-1-Phosphate Receptor-1 Promotes Environment-Mediated and Acquired Chemoresistance

doi: 10.1158/1535-7163.MCT-17-0379

Figure Lengend Snippet: (A) Western blotting showing the effects of FTY720 on pSTAT3 in CHLA-255 cells. CHLA-255 cells were serum starved for 2 h, treated for 2 h with 10 µM FTY720, and followed by 20 min incubation with BM-MSC CM (10%). (B) Western blotting comparing the indicated protein levels in control or S1pr1 overexpressing CHLA-255 cells treated with indicated concentrations of FTY720. (C) Effects of etoposide or FTY720 alone or in combination on the viability of CHLA-255 cells by MTS assay following 48 h treatment. Data represent mean ± SEM percentage of viable cells from 4 independent experiments. (D) Western blotting of indicated proteins in CHLA-255 cells treated with 5 µM FTY720 or 1 µM etoposide alone or in combination for 24 h. In panel A, B and D, data are representative of 2–3 independent experiments with similar results. (E) Immunofluorescence staining of CHLA-255+hBM-MSCs tumors, collected at the end of the in vivo experiment, with anti-CD90 (green), anti-CD44 (red) and anti-CD31 (purple) antibodies. Zoomed inset highlights cells expressing MSC markers, CD90+/CD44+/CD31− (20× magnification). (F) Relative tumor volume of NB xenografts in mice treated with vehicle (n =4), FTY720 alone (n = 6), etoposide alone (n = 5), or FTY720 plus etoposide (n = 6). Mice were treated with 5 mg/kg FTY720 daily, 5 days a week for a total of 2 weeks, 5 mg/kg etoposide on the first day of treatment and then 2 more times at 6-day interval (for a total of 3 doses), or a combination of FTY720 and etoposide. (G) Representative immunohistochemistry images for cleaved caspase-3 staining in tumor sections (20× magnification). Right panel, quantification of cleaved caspase-3-positive cells in tumor tissue. Data were analyzed by ImageJ software from 3–4 fields examined in 3 sections for each tumor.

Article Snippet: Rabbit polyclonal antibodies against pSTAT3 (Y705) (#9131), pJAK2 (#3771), cleaved PARP-1 (#9542), cleaved caspase-3 (9664), Bcl- X L (sc-8392), Bcl-2 (#2876) and survivin (#2808) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Incubation, MTS Assay, Immunofluorescence, Staining, In Vivo, Expressing, Immunohistochemistry, Software

Journal: Molecular cancer therapeutics

Article Title: Sphingosine-1-Phosphate Receptor-1 Promotes Environment-Mediated and Acquired Chemoresistance

doi: 10.1158/1535-7163.MCT-17-0379

Figure Lengend Snippet: (A) Dose dependent cell viability curve of CHLA-255 etoposide-sensitive and etoposide-resistant NB cells treated with etoposide for 24 h. The IC50 values were determined using GraphPad Prism 6 software. (B) Flow cytometry analysis of apoptosis by Annexin-V staining of the CHLA-255 and CHLA-171 etoposide-sensitive and etoposide-resistant NB cells after treatment with etoposide (1 µM) for 24 h. Data represent mean ± SEM percentage of Annexin-V positive cells of 4 independent experiments, each performed in duplicate. (C) Caspase-3/7 activation in CHLA-255 etoposide-sensitive and resistant cells responding to etoposide treatment. (D) Western blotting of S1PR1, full-length PARP-1 and its cleavage fragment, pSTAT3 and total STAT3 in CHLA-255 and CHLA-171 sensitive (S) and resistant (R) cells following exposure to etoposide for 24 h. Data are representative of 3 independent experiments with similar results. (E) Immunofluorescent (red) staining of S1PR1 in CHLA-255 sensitive and resistant cells by confocal microscopy. Nuclei were stained with Hoechst 33342. Images are representative of 3 independent experiments. Scale bar, 10 µm. Right panel, quantitation of fluorescence of immunostained cells using ImageJ. The data represent the mean + SEM total cellular fluorescence value (in pixels) from 20–25 cells analyzed.

Article Snippet: Rabbit polyclonal antibodies against pSTAT3 (Y705) (#9131), pJAK2 (#3771), cleaved PARP-1 (#9542), cleaved caspase-3 (9664), Bcl- X L (sc-8392), Bcl-2 (#2876) and survivin (#2808) were obtained from Cell Signaling Technology.

Techniques: Software, Flow Cytometry, Staining, Activation Assay, Western Blot, Confocal Microscopy, Quantitation Assay, Fluorescence

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC

doi: 10.18632/oncotarget.21066

Figure Lengend Snippet: (A) Mean fold change in percent of BrdU-positive NBT2 cells treated with solvent or JAK1/2 inhibitors AZD1480 (AZD) or ruxolitinib at indicated concentrations for NBT2 cells either alone or in co-culture with macrophages. Data were compiled from at least 3 independent experiments in triplicates; (B) Effects of AZD1480 and ruxolinitib on pSTAT3, STAT3, and MYC protein levels in lysates of NBT2 cells without or with a 6 and 24 hour co-culture with syngeneic macrophages in presence of AZD1480 (5 μM) or ruxolitinib (1 μM) as assessed by immunoblotting; (C) Immunoblot analysis of pSTAT3, STAT3, and MYC levels in protein lysates of three low MYC-expressing human NBL cell lines (LAN-6, CHLA-172, and CHLA-79) after 24 hour co-cultures with macrophages without or with ruxolitinib (1 μM); (D) Immunoblot analysis of STAT3 and pSTAT3 levels in protein lysates of tumors from NB-Tag mice or of subcutaneous tumors (Sub-Q) growing in NSG mice treated with ruxolitinib (60 mg/kg) by oral gavage twice daily for one week. (E) Tumor growth in NSG mice injected subcutaneously with 1X10 6 NBT2 tumor cells alone in one shoulder or co-injected in the opposite shoulder with equal number of macrophages that were conditioned for 24-36 hours with NBT2 cells in the transwell system. Animals were administered ruxolitinib (60 mg/kg) or drug vehicle by oral gavage twice daily for 3 weeks [ANOVA p < 0.005 between the untreated group (n = 12) and the treated group (n = 6); no significant difference was observed between treatment groups].

Article Snippet: Immunoblotting : Blots were probed with rabbit polyclonal Ab against pSTAT3 (Tyr705) (pAb #9131) and mAb against STAT3 (clone 79D7) (Cell Signaling Technology, Danvers, MA) or mAb against c-MYC (clone Y69) (Abcam, Cambridge, UK), and detected using donkey anti-rabbit IRDye fluorescently-labeled secondary Ab (LI-COR Biosciences, Lincoln, NE).

Techniques: Solvent, Co-Culture Assay, Western Blot, Expressing, Injection

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC

doi: 10.18632/oncotarget.21066

Figure Lengend Snippet: (A) Immunoblot analysis showing STAT3 expression and phosphorylated STAT3 (pSTAT3) levels over time in protein lysates of NBT2 cells cultured in transwells with and without syngeneic murine macrophages. GAPDH is used as control for protein loading; (B) STAT3 expression and pSTAT3 levels assessed by immunoblotting protein lysates from adrenal glands of WT, NB-Tag, and NB-Tag- IL6 KO mice (14-22 weeks of age); (C) Representative images of pSTAT3 IHC in tumors of NB-Tag and NB-Tag- IL6 KO mice (inset: WT adrenal gland); (D) Immunoblots of STAT3 and pSTAT3 levels in NBT2 (murine) and CHLA-255 (human) NBL cells at basal level, and in the presence of IL-6 (10 ng/ml) or sIL-6R (25 ng/ml) either alone or with macrophages previously conditioned with tumor cell media, and incubated with IgG (control) or species-specific neutralizing anti-IL-6 mAb (1 and 5 μg/ml).

Article Snippet: Immunoblotting : Blots were probed with rabbit polyclonal Ab against pSTAT3 (Tyr705) (pAb #9131) and mAb against STAT3 (clone 79D7) (Cell Signaling Technology, Danvers, MA) or mAb against c-MYC (clone Y69) (Abcam, Cambridge, UK), and detected using donkey anti-rabbit IRDye fluorescently-labeled secondary Ab (LI-COR Biosciences, Lincoln, NE).

Techniques: Western Blot, Expressing, Cell Culture, Control, Incubation