Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).
Anti c fos
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-c-Fos is an antibody used to detect the c-Fos protein in biological samples. c-Fos is a transcription factor that regulates gene expression in response to various stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Anti c jun, by Cell Signaling Technology (9 mentions)
Anti erk1 2, by Cell Signaling Technology (5 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Anti p38, by Cell Signaling Technology (4 mentions)
Anti phospho jnk, by Cell Signaling Technology (4 mentions)
Anti jnk, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti p65, by Cell Signaling Technology (3 mentions)
Anti phospho p38, by Cell Signaling Technology (3 mentions)
Anti gaba, by Merck Group (3 mentions)
Anti p erk, by Cell Signaling Technology (3 mentions)
Anti phospho c fos, by Cell Signaling Technology (3 mentions)
Anti phospho c jun, by Cell Signaling Technology (3 mentions)
Anti erk, by Cell Signaling Technology (3 mentions)
Anti phospho erk, by Cell Signaling Technology (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti phospho p65, by Cell Signaling Technology (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
37 protocols using anti c fos
1
Detailed Western Blot Methodology
2
Culturing Human Liver Cancer and Kidney Cell Lines
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The human liver cancer cell line HCCLM3 was obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China). The human liver cancer cell line Hep3B and the human embryonic kidney cell line HEK293T were obtained from American Type Culture Collection (ATCC). All cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) (Corning) supplemented with 10% fetal bovine serum (FBS) (VISTECH), 100 U/ml of penicillin and 100 µg/ml of streptomycin (P/S) (Hyclone) in a humidified incubator at 37°C with 5% CO2. Commercially available antibodies used in this study included anti-Rictor (Bethyl, A500-002A), anti-ABLIM1 (Abcam, ab222824 and Proteintech, 15129-1-AP), anti-MKL1 (Proteintech, 21166-1-AP), anti-phosphoserine (Abcam, ab9332), anti-phospho-AKT (Ser473) (Cell Signaling Technology, 4060T), anti-AKT (Cell Signaling Technology, 4685S), anti-Flag (Sigma, F3165-2MG), anti-β-Actin (Santa Cruz, sc-69879) and anti-GAPDH (Santa Cruz, sc-47724), anti-c-Fos (Cell Signaling Technology, 2250S), anti-Arp3 (Proteintech, 13822-1-AP). Mitomycin C was from Selleck (S8146).
3
Immunofluorescence Staining of Bladder Tissue
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Excised bladders were fixed in 4% w/v paraformaldehyde dissolved in 100 mM sodium cacodylate (pH 7.4) buffer for 2 h at room temperature. Fixed tissue was cut into small pieces with a razor blade, cryoprotected, frozen, and sectioned. Cells grown on collagen-coated glass coverslips were fixed for 20 min with 4% (w/v) paraformaldehyde. Tissue or cells were incubated with primary antibodies (1:100) for 2 h at room temperature. After washing away unbound primary antibody, sections were incubated with an Alexa 488-conjugated secondary antibody (diluted 1:100), and actin was stained with rhodamine phalloidin (Cytoskeleton, PHDR1). Tissue or cells were mounted with SlowFade Diamond Antifade Reagent containing DPAI (Thermo Fisher Scientific) to stain nuclei. Images acquired by Zeiss LSM-510 confocal microscope were saved as TIFF files, and imported into Adobe Illustrator CS3. Additional antibodies included rat anti-mouse β1 integrin antibody (BD Bioscience, #550531), anti-c-fos (Cell Signaling, #2250), anti-c-jun (Cell Signaling, #9165), and anti-Ki67 (Abcam, #ab15580).
4
Osteoclast Differentiation Assay Protocol
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PPOA-N-Ac-2-Cl, purchased from ChemBridge (San Diego, CA, USA), was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to obtain a 50-mM solution. Aliquots of the solution were stored at −20 °C and diluted to the appropriate concentrations in cell culture medium immediately before use. DMSO was used as the vehicle control. Alpha-modified minimal essential medium (α-MEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bone resorption assay kit was procured from Cosmo Bio Co., Ltd., Tokyo, Japan. BCA protein assay kit was purchased from Pierce Biotechnology, (Rockford, IL, USA). Primary antibodies including anti-β-actin (Sigma-Aldrich, St Louis, MO, USA), anti-c-Src (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-cathepsin K (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-ERK1/2, anti-phospho-ERK1/2, anti-AKT, anti-phospho-AKT, anti-IκBa, anti-phospho-IκBa, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-c-fos, anti-NFATc1, anti-p65, and anti-phospho-p65, anti-TRAF6, anti-calcineurinA, anti-calmodulin were purchased from Cell Signaling Technology (Boston, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology, and chemiluminescence signals were detected using an ECL system (iNtRON, Seoul, Korea).
5
Western Blot Analysis of Signaling Proteins
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After treatment with Aspergillus protease and washing with PBS, BEAS2B cells were lysed in RIPA buffer (LPS Solution, Daejeon, Korea). The cell lysates (25 μg/lane) were separated by electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with blocking solution for 50 min at 23°C. After blocking, the membranes were incubated with rabbit anti-JNK, anti-phospho- (p-) JNK, anti-ERK1/2, anti-p-ERK1/2, anti-c-FOS, anti-c-JUN, anti-UCP-2, and anti-β-actin antibodies (Cell Signaling Technology; 1 : 1000 dilution) with gentle agitation overnight at 4°C. After incubation with the primary antibodies, the membranes were washed three times with TBST and incubated with biotinylated goat anti-rabbit IgG antibodies (Cell Signaling Technology) for 2 h at room temperature. The membranes were washed three times with the TBST, developed using Clarity Western ECL Blotting Substrate (Bio-Rad, Hercules, CA), and visualized using an ImageQuant LAS 4000 mini (GE Healthcare Life Science, Chicago, IL).
6
Proteasome Subunit Immunoblotting Analysis
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Equal amounts of protein extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Merk, Darmstadt, Germany). I-proteasome subunits were analyzed by immuno-blotting using anti-mouse anti-β5i/LMP7, anti-β5, anti-β1i/LMP2 (Abcam, Cambridge, UK) and anti-β1, anti-β2i/MECL-1, anti-α4 (self-made), and AP1 components, anti-(p)-cJun, anti-(p)-cFos, anti-cFos, anti-(p)-ATF-2, anti-ATF-2 (Cell signaling, Leiden, Netherlands), and anti-cJun (Santa Cruz Biotechnologie, Dallas, Texas, USA) antibodies combined with a secondary polyclonal mouse-anti-rabbit-IgG antibody conjugated to horseradish peroxidase (Dianova, Hamburg, Germany). Anti-β-actin antibody (Cell signaling, Leiden, Netherlands) was used as loading control.
7
Quantifying Neural Activity Markers in Mice
8
Western Blot Analysis of Protein Markers
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Cells were homogenized briefly in 10 volumes of lysis buffer containing (in mM) 20 Tris–HCl (pH 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na4P2O7, 1 Na3VO4, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma Aldrich, St. Louis, MO, USA), 1% (v/v) Triton X-100 and 10% (v/v) glycerol. The homogenates were centrifuged twice at 20,000×g at 4 °C for 15 min, and the supernatants were retained as total protein. Protein concentrations were determined by the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, MA, USA). Western blot analysis was performed under standard conditions with specific anti-B7-H6 (1:2000; Abcam, MA, USA), anti-C-myc (1:1500, Abcam, MA, USA), anti-C-fos (1:2000, Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:2000, Cell Signaling Technology, MA, USA), and anti-GAPDH (1:4000, Sigma, St. Louis, MO, USA) antibodies and HRP-labeled goat anti-mouse/rabbit secondary antibody (1:6000, Sigma Aldrich, St. Louis, MO, USA). The immunoreaction was visualized using an enhanced chemiluminescence detection kit (Thermo Fisher, MA, USA) and exposure to X-ray film, and band densities were quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad).
9
ESCC Cell Lines and Antibody Acquisition
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PT and Cisplatin (DDP) were purchased from Selleckchem (Houston, TX, USA). Tumor necrosis factor-α (TNF-α) was obtained from Sigma Aldrich (St. Louis, MO, USA). Anti-CD31, anti-Ki67, anti-c-Fos, anti-c-Jun, and anti-NF-κB antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-VEGF and anti-GAPDH antibodies were obtained from Abclonal Technology (Wuhan, Hubei, China). The human ESCC lines–Eca109, KYSE-510 and human esophageal epithelial cells Het-1A were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were obtained from Xiangya Central Experiment Laboratory. The cells were cultured at 37°C in a 5% CO2 incubator using Roswell Park Memorial Institute‐1640 (RPMI‐1640, Basal Media, China) containing 10% fetal bovine serum (FBS, Biological Industries, Israel), 100 U/mL penicillin and 100 μg/mL streptomycin.
10
Immunohistochemical Analysis of Visual Cortex
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Mice were perfused transcardially with PBS followed by 4% paraformaldehyde prepared in PBS. Brains were post-fixed 1 h at 4°C and immunohistochemistry was performed on cryosections (20 μm) encompassing the entire visual cortex. Primary antibodies included anti-Myc (rabbit, 1/400, Sigma-Aldrich C3956), anti-Otx2 (mouse monoclonal, in house), anti-PV (rabbit, 1/500, Swant PV25), anti-CR (mouse monoclonal, 1/500, Swant), anti-GABA (rabbit, 1/300, Sigma), and anti-cFos (rabbit monoclonal, 1/300, Cell Signaling). Secondary antibodies were Alexa Fluor-conjugated (Molecular Probes). Biotinylated WFA (1/100, Sigma-Aldrich L1516) with Alexa Fluor-conjugated streptavidin was used to reveal perineuronal nets. Sections were mounted in Fluoromount (Southern Biotech). Images were acquired with a Leica SP5 confocal microscope and analyzed with ImageJ.
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