We performed western blotting according to a standard protocol. Total cell lysates were extracted using 2% SDS lysis buffer with a protein phosphatase inhibitor mix (Applygen, Beijing), and 30 μg of total protein was prepared for electrophoresis through 8% or 12% (v/v) SDS–PAGE gels. After electrophoresis, the separated protein bands were transferred onto nitrocellulose membranes (Millipore, Ireland) and were blocked in 5% (w/v) Albumin Bovine-V (BSA-V, Solarbio, Beijing) for 1.5 h at room temperature. The primary antibodies used were as follows: anti-GAPDH (FL-335; Santa Cruz Biotechnology), anti-CD59 (HPA026494, Sigma-Aldrich), anti-CD163 (ab182422, Abcam), anti-arginase 1(Arg-1) (16001-1-AP, Proteintech), anti-IFN-γ (ab9657, Abcam), anti-iNOS (ab3523, Abcam), anti-IL-6 (ab6672, Abcam), anti-STAT3 (D1B2J, Cell Signaling Technology), and anti-phospho-Stat3 (Ser727, Cell Signaling Technology). The quality of loading and transferring was assessed by immunostaining with the anti-GAPDH antibody. The membranes were incubated with the primary antibodies at a dilution ratio of 1:1000 overnight at 4 °C. After washing three times in TBST, the membranes were incubated in 1:5000 HRP-conjugated secondary antibodies (Zsbio, Beijing) at room temperature for 1 h. Finally, the membranes were washed three times in TBST and visualized using an ECL Kit (Applygen, Beijing).
Anti phospho stat3 ser727
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-STAT3 (Ser727) is a laboratory reagent that detects the phosphorylation of the serine 727 residue on the Signal Transducer and Activator of Transcription 3 (STAT3) protein. STAT3 is a transcription factor involved in various cellular processes. This antibody can be used to study the activation and regulation of STAT3 signaling pathways.
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Lab products found in correlation
Anti stat3, by Cell Signaling Technology (3 mentions)
Anti phospho stat3 tyr705, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ab6672, by Abcam (1 mentions)
Ab3523, by Abcam (1 mentions)
Ab9657, by Abcam (1 mentions)
Ab182422, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Albumin bovine 5 bsa 5, by Solarbio (1 mentions)
Anti gapdh fl 335, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Ecl kit, by Applygen (1 mentions)
Protein assay kit, by Solarbio (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
Bicinchoninic acid (bca), by Solarbio (1 mentions)
Anti mouse and anti rabbit secondary antibodies, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Lysis buffer, by Solarbio (1 mentions)
Anti β actin antibody, by ABclonal (1 mentions)
9 protocols using anti phospho stat3 ser727
1
Western Blotting for Protein Analysis
2
Protein Extraction and Western Blot Analysis
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Cells were resuspended in RIPA (radio-immunoprecipitation assay) lysis buffer (Solarbio, Beijing, China) supplemented with protease inhibitor cocktail (Selleck, Darmstadt, Germany). Then, cells were sonicated for 2 min, followed by centrifugation to collect the supernatant. Protein concentrations were measured using a BCA (bicinchoninic acid) protein assay kit (Solarbio, Beijing, China). Equal amounts of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto PVDF (polyvinylidene fluoride) membranes (Millipore, Billerica, MA, USA) and subjected to standard Western blot analysis. Anti-JNK, anti-phospho-JNK (Thr183/Tyr185), anti-STAT3, and anti-phospho-STAT3 (Ser727) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-caspase-3 and anti-caspase-3 (activated) antibodies were purchased from Sangon Biotech (Shanghai, China). Anti-β-actin antibody was purchased from ABclonal Technology (Wuhan, China). Anti-mouse and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Except for the Anti-β-actin antibody, which was diluted in 1:10,000, all antibodies were diluted in 1:2000.
3
Protein Extraction and Western Blot Analysis
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For total protein extracts, tissues were homogenized in protein lysis buffer and centrifuged at 15,000g during 15 minutes at 4 °C. Nuclear extracts were isolated as previously described.24 (link) The primary antibodies used were: Anti-Sirt1 (Millipore 07-131, Billerica, MA), Anti-phospho-ACC-Ser79 (Millipore 07-303), Anti-AMPK (Cell Signalling 2532, Danvers, MA), Anti-phospho-AMPK-Thr172 (Cell Signalling 2531), Anti-phospho-STAT3-Ser727 (Cell Signalling 9134), Anti-ERK1/2 (Cell Signalling 9102), Anti-phospho-ERK1/2-Thr202/Tyr204 (Cell Signalling 9101), Anti-Acetyl-p53-Lys379 (Cell Signalling 2570), Anti-PGC1 (Santa Cruz H-300, Dallas, TX), Anti-α-tubulin (Abcam ab4074, Cambridge, UK). Detection was performed using the corresponding horseradish peroxidase-labeled secondary antibodies and Western blotting detection reagent (ECL Plus; Amersham, Freiburg, Germany).
4
ChIP-Seq Analysis of Transcription Factors
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Chromatin immunoprecipitation (ChIP) analysis was performed for two biological replicates using a ChIP kit (Millipore Magna ChIP A/G) according to the manufacturer’s protocol. Briefly, cells were cultivated in 150-mm plates to 90% confluence. The cells were fixed for 10 min in 1% freshly made formaldehyde, washed with phosphate-buffered saline (PBS), and lysed for 15 min on ice in lysis buffer provided in the kit. Chromatin was sheared by sonication to an average size of ~200 to 500 bp and clarified. Chromatin was incubated with normal rabbit immunoglobulin G, with anti-bcl3 (sc-185x; Santa Cruz), anti-p50 (ab7971-1; Abcam), or anti-phospho-STAT3 (Ser727) (Cell Signaling 9134) at 4°C overnight on a nutator. Protein A/G magnetic beads were added, and the samples were incubated at 4°C for 2 h on a nutator. The antibody/DNA/magnetic bead complexes were thoroughly washed, the protein/DNA complexes were eluted, and the cross-linking was reversed by proteinase K digestion for 2 h at 62°C to free the DNA. Sample DNAs were purified for further analysis according to the manufacturer’s protocol. For ChIP-seq samples, DNA from ChIP was submitted to the University of North Carolina High Throughput Sequencing Facility for library preparation and high-throughput sequencing. Single-end 50-bp sequences were generated from each library using the Illumina HiSeq 2000 platform.
5
Western Blotting Analysis of Signaling Pathways
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For Western blotting analysis, cells were lysed as previously described [63 (link), 64 (link)]. Protein determination was performed by BCA Protein Assay (Thermo Scientific, Rockford, IL). Equal amounts of protein for each sample were migrated in SDS-polyacrylamide gels and blotted onto nitrocellulose filters. The following Abs were used: anti-Mcl-1 (S-19) and anti-Bcl-2 (100) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-AMPKα (Thr172, D79.5E), anti-AMPKα, anti-phospho-STAT3 (Ser727) and anti- STAT3 from Cell Signalling (Danvers, MA); anti-phospho-Akt1/PKBα (Ser473) from Merck Millipore (Darmstadt, Germany); anti AKT/PKBα from BD; anti-tubulin from Sigma-Aldrich. After incubation with anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary Abs (Sigma-Aldrich), specific reactions were revealed with the ECL Lightning detection kit (Perkin Elmer, Waltham, MA). The estimation of the densitometry values of bands was obtained by the ImageQuant TL software (GE Healthcare, Buckinghamshire, UK).
6
Protein Expression and Phosphorylation Analysis
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To determine protein expression and phosphorylation status, transfected A549 cells with empty vector pcDNA3.1 or pSARS-PLpro were harvested 2 days after transfection. Western blotting of cell lysates was accomplished, as described in our prior reports12 (link)13 (link). Resulting blots were probed with primary antibodies, including mouse polyclonal anti-E. coli synthesized PLpro, rabbit anti-vimentin (GeneTex), rabbit anti-TGF-β1 (Cell signaling), anti-α-SMA (Santa Cruz Biotechnology), rabbit anti-Egr-1, anti-phospho Erk1/2 (Thr202/Tyr204), anti-phospho p38 MAPK (Thr180/Tyr182), anti-phospho STAT3 (Ser727) (Cell Signaling), and anti-β-actin mAb (Abcam). Immune complexes were detected using HRP-conjugated goat anti-mouse or anti-rabbit IgG antibodies, as well as enhanced chemiluminescent HRP substrate (Millipore).
7
Western Blot Analysis of DYRK1A and STAT3
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Cells or wound tissues were homogenized in radioimmunoprecipitation assay buffer (P0013B, Beyotime Biotechnology, China), and protein concentrations were determined by bicinchoninic acid protein assay (P0010, Beyotime Biotechnology). Equal amounts of protein were loaded, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked in 5% skim milk at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies. The membranes were then extensively washed in Tris-buffered saline with 0.1% Tween® 20 detergent (TBST) and incubated with secondary antibodies for 2 h at room temperature. After being washed three times with TBST, protein bands were detected using an enhanced chemiluminescence detection system (Bio-Rad Laboratories, Inc.). Luminescence intensity was analyzed using ImageJ. The antibodies used were anti-DYRK1A (YT1435, Immunoway, USA), anti- phospho-STAT3 (Tyr705) (9145S, Cell Signaling Technology, USA), anti- phospho-STAT3 (Ser727) (9134S, Cell Signaling Technology, USA), anti-STAT3 (9139S, Cell Signaling Technology, USA), anti-β-actin (AF7018, Affinity Biosciences, China), and anti-rabbit IgG (A7016, Beyotime Biotechnology, China).
8
Immunoblotting Analysis of STAT3 Signaling
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Cells were lysed in NET buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA pH 8) and immunoblotted with the following antibodies: anti-STAT3 (#9139), anti-phospho-STAT3(Tyr705) (#9131), anti-phospho-STAT3(Ser727) (#9134), anti-IL1β (#12242), anti-phospho-JAK2(Tyr1007/1008) (#3771), anti-PAI-1 (D9C4) (#11907) from Cell Signaling; anti-NFκB p65 (sc-372), anti-USP18 (sc-98431) and anti-Actin (sc-1616) from Santa Cruz Biotechnology. Proteins of interest were detected with HRP-conjugated anti-mouse/rabbit/goat IgG antibodies from Santa Cruz Biotechnology and visualized with the Pierce ECL Western blotting substrate (ThermoScientific), according to the provided protocol. Autoradiography images were developed with a KODAK MIN-R processor. Full-length blots are included in Supplementary Figures 9/10 .
9
Characterization of Multiple Myeloma Cell Lines
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Reagents and antibodies. Recombinant human CX3CL1 and anti-human CX3CL1 were purchased from R&d Systems (Minneapolis, MN, uSA). The following mAbs were used: anti-AKT1 (C-20), anti-ERK1/2 (C-16), anti-PCNA (PC-10) and anti-NFkbp65 (C-20) from Santa Cruz biotechnology (Santa Cruz, CA, uSA), and anti-phospho AKT (Ser-473), anti-phospho p44/42 ERK (Thr-202/Tyr-204), anti-STAT3 and anti-phospho STAT3 (Ser727) from Cell Signaling Technology (beverly, MA, uSA).
Cells. Human multiple myeloma cell lines included RPMI-8226, KMS-12bM, KMS-12PE, L-363, OPM-2, KARPAS-620 and AMO-1 cells. All the cell lines were maintained in RPMI-1640 medium (Wako Pure Chemical Industries, Inc., Osaka, Japan) supplemented with 20% fetal bovine serum, 50 µM 2-mercaptoethanol (both from Invitrogen, Carlsbad, CA, uSA), 100 u/ml penicillin and 100 µg/ml streptomycin (both from Meiji Seika Pharma, Tokyo, Japan). The cells were cultured at 37˚C in an incubator with a humidified 5% CO 2 atmosphere.
Cells. Human multiple myeloma cell lines included RPMI-8226, KMS-12bM, KMS-12PE, L-363, OPM-2, KARPAS-620 and AMO-1 cells. All the cell lines were maintained in RPMI-1640 medium (Wako Pure Chemical Industries, Inc., Osaka, Japan) supplemented with 20% fetal bovine serum, 50 µM 2-mercaptoethanol (both from Invitrogen, Carlsbad, CA, uSA), 100 u/ml penicillin and 100 µg/ml streptomycin (both from Meiji Seika Pharma, Tokyo, Japan). The cells were cultured at 37˚C in an incubator with a humidified 5% CO 2 atmosphere.
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