Immunofluorescences (IF) and immunoblots (WB) were performed with the following primary antibodies: anti-rabbit S100A4 (1:500-IF, 1:1000-WB, Millipore), anti-rabbit mTOR and phospho-mTOR (1:100-WB, Cell Signaling), anti-mouse SQSTM1/p62 (1:1000-WB, Abcam), anti-rabbit NF-kB and phospho-NF-kB (1:1000-WB, Cell Signaling), anti-rabbit STAT3 (1:1000-WB, Cell Signaling), anti-rabbit phospho-STAT3 (1:2000-WB, Cell Signaling), anti-rabbit α-SMA (1:1000-WB, 1:500-IF, GeneTex), anti-rabbit N-cadherin (1:1000-WB, GeneTex), anti-mouse glial fibrillary acidic protein (GFAP) (1:500-IF, 1:1000-WB, Cell Signaling), anti-rabbit β-III Tubulin (1:500-IF, Cell Signaling), anti-mouse MyoG (1:200-WB, Hybridoma Bank, USA), anti-mouse platelet-derived growth factor receptor β (PDGFR-β) (1:250-WB, 1:500-IF, Santa Cruz), and anti-mouse GAPDH (1:10000-WB, Calbiochem). Secondary immunoblot antibodies for WB were anti-rabbit (1:2500) and anti-mouse (1:5000) IgG peroxidase-conjugated from Bio-Rad Laboratories (Hercules, CA, USA). Secondary fluorescent antibodies for IF were Alexa-Flour 488-Donkey anti-rabbit (1:200) and Cy3-Donkey anti-mouse (1:200) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). DAPI was used to stain nuclei (1:1000, Thermo Fisher Scientific, Waltham, MA, USA).
Rabbit anti stat3
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Rabbit anti-STAT3 is a primary antibody that recognizes the Signal Transducer and Activator of Transcription 3 (STAT3) protein. STAT3 is a transcription factor that plays a key role in cellular processes such as cell growth, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti pstat3, by Cell Signaling Technology (12 mentions)
Rabbit anti phospho stat3, by Cell Signaling Technology (10 mentions)
Rabbit anti β actin, by Cell Signaling Technology (4 mentions)
Rabbit anti jak2, by Cell Signaling Technology (4 mentions)
Rabbit anti phospho stat3 tyr705, by Cell Signaling Technology (4 mentions)
Rabbit anti akt, by Cell Signaling Technology (3 mentions)
Mouse anti β actin, by Merck Group (3 mentions)
Rabbit anti pstat3, by Abcam (3 mentions)
Rabbit anti p38, by Cell Signaling Technology (3 mentions)
Mouse anti stat1, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rabbit anti stat1, by Cell Signaling Technology (3 mentions)
Rabbit anti phospho jak2, by Cell Signaling Technology (2 mentions)
Rabbit anti mmp 2, by Cell Signaling Technology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Goat anti iba1, by Fujifilm (2 mentions)
Rabbit anti cox 2, by Abcam (2 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Rabbit anti il 1β, by Abcam (2 mentions)
Rabbit anti erk, by Santa Cruz Biotechnology (2 mentions)
53 protocols using rabbit anti stat3
1
Immunofluorescence and Immunoblotting Analysis
2
Oridonin Modulates EGFR Signaling Pathway
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In order to evaluate the effect of oridonin on EGFR pathway proteins and validate the in silico results, varying concentrations of oridonin (IC50/4, IC50/2, IC50, 2xIC50, and 4xIC50), determined after the cytotoxicity test on U87MG.ΔEGFR cell line, were applied in a similar way as described previously (Saeed et al., 2015 (link)). Briefly, 1 million cells per well were seeded in 12-well plate, next day treatment with oridonin was performed, total protein were extracted after 24 h. The following primary antibodies (Cell Signaling Technology, Frankfurt, Germany) were used: anti-rabbit EGFR, anti-rabbit phosphorylated EGFR (Tyr1068) (1:1000), anti-rabbit STAT3, anti-mouse phosphorylated STAT3 (Tyr705) (1:1000), anti-rabbit Akt, anti-rabbit phosphorylated Akt (Ser473) (1:1000), and anti-rabbit β-actin (1:2000).
3
Antibody-based Assay for MMP2, MMP9, and Signaling Pathways
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Anti-rabbit matrix metalloproteinase (MMP) 2, MMP9 monoclonal antibodies were purchased from Epitomics Inc., Abcam (Cambridge, MA, USA). Anti-rabbit STAT3, p-STAT3, vascular endothelial growth factor (VEGF) and suppressor of cytokine signaling 1 (SOCS1) polyclonal antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); MTT was obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA); fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) culture medium were purchased from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA) and Transwell Chambers from Corning Incorporated (Corning, NY, USA); mini double vertical electrophoresis apparatus, mini transfer electrophoresis apparatus, ChemiDoc™ XRS Gel Imaging System were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA); and TE2000 fluorescence inverted microscope was obtained from Nikon Corporation (Tokyo, Japan).
4
THSG Modulates JAK2/STAT3 Signaling
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After the cells were treated with 0, 1, 10, and 100 μM THSG in PluriSTEMTM ES/iPS Medium (Merk) for 3 h, protein extraction and Western blotting were performed as described previously.13 (link) The resolved proteins were then incubated with rabbit anti-JAK2, rabbit anti-phospho-JAK2, rabbit anti-STAT3, rabbit anti-phospho-STAT3 (Cell Signaling Technology, Beverly, MA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Merk, Darmstadt, Germany) at 4 °C overnight. Signals were detected using the LumiFlas™ Femto Chemiluminescent Substrate, HRP kit (ENERGENESIS BIOMEDICAL, Taipei, Taiwan). Images of the Western blots were visualized and analyzed using the Chemi-Doc™ XRS + System (Bio-Rad Laboratories, Hercules, CA, USA).
5
Multiplex Cytokine Immunodetection
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Rabbit anti-actin (Sigma), rat anti-IL1-beta/IL-1F2 (R&D), rabbit anti-TGF-beta (Nordic Biosite), rat anti-GM-CSF (Abcam), rat anti-IL10 (Abcam), goat anti-M-CSF (Abcam), goat anti-VEGFD (Abcam), rabbit anti-VEGF (Abcam), rabbit anti-VEGFC (Abcam), rabbit anti-PlGF (Abcam), rabbit anti-IL-6 (Abcam), rabbit anti-phospho (Ser727) STAT3 (Cell Signaling), rabbit anti-phospho, (Tyr705) STAT3 (Cell Signaling), rabbit anti-STAT3 (Cell Signaling). Anti-GM-CSF, IL-10 and IL-1beta were monoclonal antibodies, all other antibodies were polyclonal.
6
Investigating Cytokine and Signaling Pathways in EMT
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Human recombinant IL-6 and IL-6 neutralizing antibody were purchased from R&D Systems Inc. (Minneapolis, MN, USA). STAT3 inhibitor Stattic was purchased from Sigma-Aldrich Inc. (St Louis, MO, USA), JAK2 inhibitor AG490 was purchased from Abcam (Cambridge, MA, USA).
Antibodies used for western blotting were mouse anti-E-cadherin (Invitrogen, Carlsbad, CA, USA), mouse anti-vimentin (BD Bioscience, San Jose, CA, USA), rabbit anti-phospho-JAK2 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-phospho-STAT3 (Cell Signaling Technology), mouse anti-α-SMA (Abcam), rabbit anti-MMP2 (Cell Signaling Technology), mouse anti-VEGF (Cell Signaling Technology), rabbit anti-TWIST (Cell Signaling Technology), mouse anti-β-actin (Sigma-Aldrich). The secondary antibodies coupled to HRP were purchased from ZSGB-BIO (Beijing, China)
Antibodies used for western blotting were mouse anti-E-cadherin (Invitrogen, Carlsbad, CA, USA), mouse anti-vimentin (BD Bioscience, San Jose, CA, USA), rabbit anti-phospho-JAK2 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-phospho-STAT3 (Cell Signaling Technology), mouse anti-α-SMA (Abcam), rabbit anti-MMP2 (Cell Signaling Technology), mouse anti-VEGF (Cell Signaling Technology), rabbit anti-TWIST (Cell Signaling Technology), mouse anti-β-actin (Sigma-Aldrich). The secondary antibodies coupled to HRP were purchased from ZSGB-BIO (Beijing, China)
7
Western Blot Analysis of Inflammatory Signaling
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Synovial tissues from the right forepaw joints and treated macrophages were lysed in RIPA buffer (Solarbio, Beijing, China, Cat#R0020) with phenylmethylsulfonyl fluoride (Solarbio, Cat#P0100) and protein phosphatase inhibitor (Solarbio, Cat#P1260). Protein samples were separated by SDS-PAGE. Separated proteins were transferred onto polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA, Cat#IPVH00010). For specific protein detection, the following primary antibodies were used: rabbit anti-STAT3 (Cell Signaling Technology, Danvers MA, USA, Cat#9139S), rabbit anti-Phospho-STAT3 (Cell Signaling Technology, Cat#9145S), rabbit anti-NLRP3 (Invitrogen, Waltham, MA, USA, Cat# PA5-79740), mouse anti-HIF-1α (Santa Cruze, Dallas, TX, USA, Cat#13515), and rabbit anti-GAPDH (Abcam, Cambridge, UK, Cat# 8245). Lastly, the Western blotting bands were analyzed with Image J Software.
8
Immunochemical Analysis of Neuroinflammation
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The following primary antibodies were used throughout this study: rat anti-mouse CD11b (1:400, Abcam), rabbit anti-F-actin (1:1000, Abcam), rabbit anti-COX-2 (1:1000, Abcam), rabbit anti-IL-1β (1:200, Abcam), rabbit anti-GFAP (1:5000, Neuromics), rabbit anti-Iba-1 (1:1000, Wako), goat anti-Iba-1 (1:500, Wako), rabbit anti-AKT (1:1000, Santa Cruz), rabbit anti-p-AKT (Ser473, Thr308) (1:1000, Cell Signaling), rabbit anti-ERK (1:1000, Santa Cruz), rabbit anti-p-ERK (Thr42/44) (1:1000, Cell Signaling), rabbit anti-STAT3 (1:1000, Cell Signaling), rabbit anti-p-STAT3 (Ser727, Abcam), mouse anti-PCNA (1:1000, Santa Cruz), rabbit anti-D2R (1:1000, Abcam), and rabbit anti-D1R (1:1000, Millipore) antibodies. We used the following small molecules: D1R antagonists (LE300, 10 μM, Sigma-Aldrich; SCH23390, 30 μM, Tocris), D1R agonist (A77636 hydrochloride, 10 nM, Tocris), D2R antagonist (eticlopride hydrochloride, 100 nM, Sigma-Aldrich), a STAT3 inhibitor (S3I-201, 50 μM, Sigma-Aldrich), and an ERK inhibitor (PD98059, 10 μM, Millipore).
9
Western Blot Analysis of STAT3 Activation
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Cells or tumor tissues were lysed in RIPA lyzing buffer (Beyotime, China), followed by centrifugation at 12,000 rpm and 4◦C for 10 min. The supernatant was harvested and the concentration of protein was measured using a BCA protein assay kit (Thermo Fisher Scientific). The protein samples were electrophoresed in 10% SDS-PAGE gel and electro-transferred to a PVDF membrane. After blocking in TBST containing 5% (w/v) BSA, the membrane was incubated with rabbit anti-STAT3, anti-p-STAT3, or anti-GADPH (1:1000, all from Cell Signaling Technology) antibody at 4°C overnight. The membrane was then incubated with HPR-conjugated goat anti-rabbit IgG (1:2000, Cell Signaling Technology) for 1 h at room temperature. Finally, blots were detected by a Bio-Rad Gel imaging system and analyzed using Image J software.
10
Antibody-Based Analysis of Signaling Pathways
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The antibodies used in this study included the following: anti-IL-6 neutralizing antibody (R&D systems), rabbit anti-JAK2, rabbit anti-phospho (Tyr1007-10008)-JAK2, rabbit anti-STAT3, rabbit anti-phospho (Tyr705)-STAT3, rabbit anti-Vimentin, rabbit anti-Snail (Cell Signaling); mouse anti-Twist, mouse anti-MMP9, goat anti-β-actin, mouse anti-HSC70 (Santa Cruz); peroxidase affinipure anti-mouse IgG and peroxidase affinipure anti-rabbit IgG (Jackson ImmunoResearch).
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