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Phospho c jun ser63

Manufactured by Cell Signaling Technology
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Phospho-c-Jun (Ser63) is a laboratory reagent used to detect and quantify the phosphorylation of c-Jun at serine 63. c-Jun is a transcription factor that plays a key role in cellular signaling pathways. The phosphorylation of c-Jun at serine 63 is a crucial regulatory event that modulates its activity and function.

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18 protocols using phospho c jun ser63

1

Antibody Detection Assays

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The antibodies against the following proteins were used: human ERα (Santa Cruz Biotechnology, #sc-543); HA (hybridoma clone 12CA5; kindly provided by Peter Angel, DKFZ, Germany); Rb (BD Biosciences, #554136); c-Jun (Cell Signaling Technology, #9165), phospho-c-Jun (Ser63) (Cell Signaling Technology, #9261), myc Tag (Upstate, #06-549); E1A (Calbiochem, #DP11-100UG; Santa Cruz Biotechnology, #sc-58658); RhoA (Cytoskeleton, #ARH03), Rac1 (Upstate, #05-389); Cdc42 (Thermo Scientific, #89857D), Ezrin (Cell Signaling Technology, #3145), Radixin (Cell Signaling Technology, #2636), Moesin (Cell Signaling Technology, #3150).
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2

Cytokine-mediated Signaling Pathway Activation

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Recombinant human IL-33 and recombinant human IL-1β were purchased from PeproTech (Rocky Hill, NJ). The mouse monoclonal anti-human IL-8 antibody, recombinant human ST2/IL-33R Fc Chimera, and the mouse IgG isotype control were from R&D System (Minneapolis, MN). The rabbit polyclonal antibodies specific for JNK, phospho-JNK (Thr183/Tyr185), c-Jun, and phospho-c-Jun (Ser63) were obtained from Cell Signaling Technology (Beverly, MA). SP600125 was purchased from BIOMOL (Plymouth Meeting, PA).
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3

Antibody Reagents for Stem Cell Research

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Antibodies against Sox2 (#3579), Nanog (#4903), Bmi1 (#6964), E-cadherin (#3195, for immunocytochemistry), glucocorticoid receptor (#12041), phospho-glucocorticoid receptor (Ser-211) (#4161), c-Jun (#9165), phospho-c-Jun (Ser-63) (#9261), phospho-SAPK/JNK (Thr-183/Tyr-185) (#4668), survivin (#2808), GFAP (#3670), phospho-Smad2 (Ser-465/467) (#3108), Smad2/3 (#8685), and GAPDH (#5174) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against JNK1 (sc-474), JNK2 (sc-7345), E-cadherin (sc-8426, for immunoblotting), and MKP-1 (sc-370) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The antibody against nestin (MAB5326) was purchased from Merck Millipore (Darmstadt, Germany). Anti-CD133 (W6B3C1) was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). Anti-β-actin (A1978) was from Sigma–Aldrich. Dexamethasone (DEX) was purchased from Fuji Pharma Co., Ltd. (Tokyo, Japan). Prednisolone (PSL), 5-FU, and gemcitabine (GEM) were from Sigma–Aldrich. DEX, PSL, 5-FU, and GEM were dissolved in PBS (for DEX and PSL) or DMSO (for 5-FU and GEM) to prepare 1, 10, 200, and 1 mm stock solutions for in vitro use, respectively.
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4

Protein Extraction and Western Blot Analysis

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Cells were harvested and homogenised in modified radioimmunoprecipitation assay buffer (modified RIPA) supplemented with 50 mM Tris‐HCl (pH 7.4), 1% NP‐40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), aprotinin (1 mg/ml), 1 mM phenylmethylsulphonyl fluoride and leupeptin (1 mg/ml). Primary antibodies against fibronectin (#15613‐1‐AP, ProteinTech), Collagen I (#14695‐1‐AP, ProteinTech), α‐SMA (GTX100904, GeneTex), phospho‐SAPK/JNK (Thr183/Tyr185) (#4668; Cell Signaling), SAPK/JNK (#9252; Cell Signaling), phospho‐NF‐κb p65 (Ser536) (#3033; Cell Signaling), NF‐κb p65 (GTX107678; GeneTex), phospho‐c‐Jun (Ser 63) (#2361, Cell Signaling), c‐Jun (#9165, Cell Signaling), phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4377; Cell Signaling), p44/42 MAPK (ERK1/2) (#9102; Cell Signaling), CD44 (ab157107; Abcam) and α‐tubulin (T6199, Sigma) were used for Western blotting. α‐tubulin was used as the loading control.
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5

Profiling PDGF-BB Signaling Pathways

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Recombinant human PDGF-BB was from R&D Systems (Minneapolis, MN). Antibodies to PDGFRα, PDGFRβ, phospho-PDGFRα/β Tyr849/Tyr857, c-Jun, phospho-c-Jun Ser63, c-Myc, EGR1, RUNX1, DDIT3, CYR61 and GDF15 were from Cell Signaling Technology (Danvers, MA); antibodies to Myb and NFAT5 were from Epitomics (Burlingame, CA); antibodies to SOX5 and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA); antibody to β-actin was from Sigma Aldrich (Sigma Chemical Company, St. Louis, MO); antibody to DIAPH3 was a generous gift from Henry Higgs, Dartmouth Medical School. The c-Myc TF ELISA kit was from Active Motif (Carlsbad, CA). SP600125 and 10048-F4 were from EMD Biosciences (Billerica, MA). iScript cDNA synthesis reagents were from BioRad Laboratories (Hercules, CA). Universal PCR master mix for qRT-PCR and gene-specific assays were from Applied Biosystems (now Life Technologies, Grand Island, NY). Primers for human transcripts were as follows: Hs00171022_m1 for CXCL12; Hs00998500_g1 for CYR61; Hs01107330_m1 for DIAPH3; Hs02758991_g1 for GAPDH; Hs00171132_m1 for GDF15; Hs01110250_m1 for HMOX-1; Hs00998018_m1 for PDGFRA; and Hs01019589_m1 for PDGFRB. Primers for mouse transcripts were Mm00487499_g1 for CYR61; Mm99999915_g1 for GAPDH; Mm00442228_m1 for GDF15; Mm00435546_m1 for Pdgfrb.
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6

Western Blot Analysis of Signaling Proteins

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Cells were lysed in cell lysis buffer (#P0013, Beyotime Biotechnology, China). In total, 20 μg of protein samples was subjected to SDS-PAGE. Next, the proteins were transferred onto PVDF membranes (#ISEQ00010, Merck Millipore, Germany). Then, the membranes were blocked in 5% skim milk for 2 h and incubated with primary antibodies overnight at 4°C. The primary antibodies for GAPDH (1:1,000, # 5,174), Smad3 (1:1,000, # 9,523T), phospho-Smad3 (Ser423/425) (1:1,000, # 9,520), c-Jun (1:1,000, # 9,165), and phospho-c-Jun (Ser63) (1:1,000, # 2,361) were purchased from Cell Signaling Technology (Danvers, MA, United States). Then, the membranes were incubated for 2 h with secondary antibodies (1:2,000, # 7074, Cell Signaling Technology) at 25°C. After the membranes were washed in TBST, chemiluminescent signals were detected using the Bio-Rad Molecular Imager ChemiDocTM XRS + system (Bio-Rad, United States). GAPDH was used as the loading control.
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7

Western Blot Analysis of Neuronal Proteins

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Frozen cerebellar tissue or frozen DRG tissue was homogenized in radioimmunoprecipitation assay buffer (RIPA) buffer containing 2× Halt protease and phosphatase inhibitor cocktail (Invitrogen, Carlsbad, CA). 35 µg of total protein was then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) using NuPAGE 4 to 12% Bis-Tris Gels (Invitrogen) and 1× MES running buffer (Invitrogen). The proteins were transferred to nitrocellulose membranes (Invitrogen). Membranes were blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE). Primary antibodies against DLK (75-355, 1:500, NeuroMab, Davis, CA), phospho-c-Jun (Ser63) (9261, 1:250, Cell Signaling Technology, Danvers, MA), c-Jun (9156, 1:250, Cell Signaling Technology, Danvers, MA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (MAB374, 1:5000, Millipore, Burlington, MA) were used, followed by LI-COR secondary antibody (1:5000). The membranes were imaged using the LI-COR Odyssey Imager and analyzed with Image Studio 4.0 software.
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8

Antibody Panel for Signaling Pathway Analysis

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The following antibodies were used: PR65 (sc-15355, Santa Cruz, CA, USA), PP2Ac (05-421, Millipore), FLAG M2 (F1804, Sigma Aldrich), HA (sc-7392, Santa Cruz), phospho-AKT (Thr308) (9275, Cell Signaling), phospho-AKT (Ser473) (9271, Cell Signaling), phospho-SRC (Tyr416) (6943, Cell Signaling), phospho-FAK (Tyr397) (3283, Cell Signaling), phospho-PP2Ac (Tyr307) (ab32104, Abcam), SRC (2108, Cell Signaling), p21 (2946, Cell Signaling), β-actin (sc-4778, Santa Cruz), phospho-SAPK/JNK (Thr183, Tyr185) (9251, Cell Signaling), phospho-c-Jun (Ser73) (9164, Cell Signaling), phospho-c-Jun (Ser63) (9261, Cell Signaling), and c-Jun (sc-1694, Santa Cruz).
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9

Elucidating ErbB2-mediated signaling cascades

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Cells were treated with 5μg/ml anti-ErbB2 antibodies for 4h at 37°C. After washing, the cells were lysed in SDS lysis buffer and the cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against ErbB2, phospho-ErbB2-Tyr1221/1222, ErbB3, phospho-ErbB3-Tyr1289, AKT, phospho-AKT-Ser473, p44/42 MAPK, phospho-p44/42 MAPK-Thr202/Tyr204, SAPK/JNK, phospho-SAPK/JNK-Thr183/Tyr185, c-Jun, phospho-c-Jun-Ser63, PARP, Caspase-3, LC3A/B, Bak, Bax, Puma, Bid, Bim, Mcl-1, Bcl-xl, phospho-Bcl-2-Ser70, Bcl-2 (all from Cell Signaling Technology).
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10

Western Blotting Procedures for Protein Detection

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Standard protocols for Western blotting were used as described previously.5 (link) Briefly, cells were lysed in standard buffers with protease inhibitors (Roche) and phosphatase inhibitors (Santa Cruz) with extracts run on sodium dodecyl sulfate (SDS)– polyacrylamide gels and transferred to an Immobilon-P transfer membrane (Millipore). Blots were blocked in Odyssey blocking buffer. Proteins were detected using the following commercially available primary antibodies. The primary antibodies against JNK1 2C6 (catalog no. 3708), JNK2 (catalog no. 4672), V5-Tag (catalog no. 13202), SAPK/JNK (catalog no. 9252), phospho-SAPK/JNK Thr183/Tyr185 (catalog no. 4668), c-Jun 60A8 (catalog no. 9165), and phospho-c-Jun Ser63 (catalog no. 9261) were purchased from Cell Signal (Beverly, MA) and used at the indicated concentrations as described by the manufacturer. Fluorescently labeled DyLight800 and DyLight680 secondary antibodies (catalog nos. 5257, 5366, 5470, and 5151; 1:5000; Cell Signaling Technology) were used, and signals were detected using the Li-COR Odyssey Fc imager (LI-COR Biosciences).
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