C fos

Manufactured by Cell Signaling Technology

Sourced in United States, China

C-Fos is a laboratory product offered by Cell Signaling Technology. It is a nuclear phosphoprotein that is rapidly and transiently induced by a variety of cellular stimuli. C-Fos is a commonly used marker for neuronal activation.

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167 protocols using c fos

Immunostaining of Synaptic Proteins

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Anti-ShhN (#2207; epitope surrounding Glu53 of human Shh) and cFos (#2250S) were from Cell Signaling Technology. Anti-ShhN (5E1) was from the Developmental Studies Hybridoma Bank. Anti-ShhC (#S9947; epitope-amino acids 199-437 of mouse Shh), anti-synaptophysin (#S5768), anti-map2 (#M9942) and anti-actin (#A5441 and #A2066) were from Sigma-Aldrich. smi312 antibody (#SMI-312R) was from Convance/BioLegend. Anti-bassoon (#141002), anti-homer1 (#160004), anti-Aldh1L1 (#278003), and anti-Gfap (#173002) were from Synaptic Systems. Anti-psd95 (#7E3-1B8) was from Thermo Fisher Scientific. Bicuculline (#0130) and PACAP (#1183) were from Tocris Bioscience. Purmorphamine (#ALX-420-045) was from Enzo Biochem Inc. Cytosine β-D-arabinofuranoside (#C1768) and Tetrodotoxin (#T5551) were from Sigma-Aldrich.

Rivell A., Petralia R.S., Wang Y.X., Clawson E., Moehl K., Mattson M.P, & Yao P.J. (2019). Sonic hedgehog expression in the postnatal brain. Biology Open, 8(3), bio040592.

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Immunofluorescence Quantification of Neuronal Activation

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For immunofluorescence, coronal brain slices were sectioned at a thickness of 30 μm, washed with phosphate buffered saline (PBS), and then incubated for blocking with permeabilization buffer (0.3% Triton‐100 in PBS) containing 10% donkey serum for 45–60 min. The sections were incubated in primary antibodies against NeuN (Millipore) and c‐Fos (Cell Signaling Technology) overnight at 4°C. The sections were rinsed three times (10 min each) in PBS, permeabilized with 0.1% Tween‐20 in PBS, and then incubated at 4°C overnight with Alexa Fluor secondary antibodies specific for the primary antibody host. Following incubation with secondary antibodies, the sections were rinsed three times (10 min each) in PBS, mounted on gelatin‐coated glass slides, and coverslipped using mounting medium. For EdU staining, we used Click‐iT^® Plus EdU Imaging Kits (Life Technologies). The images were obtained using an Olympus microscope. The analysis of immuno‐positive neurons in the hippocampal area was quantified with ImageJ software. Five brain sections were collected for quantifying the positive cells in each mouse.

Wang H., Li Q., Tang H., Ding J., Xu N., Sun S, & Chen S. (2019). The activated newborn neurons participate in enriched environment induced improvement of locomotor function in APP/PS1 mice. Brain and Behavior, 9(7), e01316.

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Immunofluorescence and Immunohistochemistry Analysis

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Transfected cells were fixed, blocked and hybridized with primary antibodies, including P-GAP43 (Abcam; 1:500 dilution), RANBP10 (Proteintech; 1:500) and β-tubulin (Sigma; 1:1,000 dilution) antibodies. The cells were then reacted with secondary antibodies conjugated with Alexa fluor 488, 594 or 647 (Invitrogen), and cellular nuclear were stained by 1μg/mL Hoechst 33342 (Sigma). Fluorescence images were captured using a DM2500 fluorescent microscope (Leica) for analyses. For 3,3'-Diaminobenzidine (DAB) immunohistochemical staining, brain sections were blocked, incubated with c-Fos (Cell signaling; 1:250) and examined with a Vectastain Elite ABC kit (Vector Laboratories) and DAB (Vector Laboratories). The intensity of c-Fos was quantitated using ImageJ (NIH), and results were subjected to statistical analyses.

Her L.S., Mao S.H., Chang C.Y., Cheng P.H., Chang Y.F., Yang H.I., Chen C.M, & Yang S.H. (2017). miR-196a Enhances Neuronal Morphology through Suppressing RANBP10 to Provide Neuroprotection in Huntington's Disease. Theranostics, 7(9), 2452-2462.

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Immunohistochemical Analysis of Wnt Signaling

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After routine deparaffinization and hydration, tissue sections were treated with 0.01 M citrate buffer (pH 6.0) for antigen retrieval. Following 3% hydrogen peroxide incubation and 10% bovine serum albumin deprivation, slides were incubated with primary antibodies of c-Fos (1:100, Cell Signaling), Wnt2 (1:100, Abcam), and Fzd9 (1:100, Abcam) at 4°C overnight. Primary antibodies were recognized by the biotinylated secondary antibody and visualized by Vectastain avidin–biotin complex peroxidase system (Zsbio, China) and 3,3′-diaminobenzidine kit (DAB, Zsbio, China). The degrees of immunoreactivity were evaluated in accordance with our previous report[14 (link)]. We performed staining by only adding phosphate-buffered saline (PBS) instead of using primary antibodies to the sections as negative controls. The sections were photographed using an optical microscope (Olympus, BX53F, Japan) and then analyzed using the Image-Pro Plus 6.0 analytic system.

Wang Q., Liu H., Wang Q., Zhou F., Liu Y., Zhang Y., Ding H., Yuan M., Li F, & Chen Y. (2017). Involvement of c-Fos in cell proliferation, migration, and invasion in osteosarcoma cells accompanied by altered expression of Wnt2 and Fzd9. PLoS ONE, 12(6), e0180558.

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EGCG-5'Glu Inhibits Cell Signaling

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EGCG-5′Glu was obtained from AmorePacific Co. (Yongin, Korea), as reported previously [32 (link)]. Cell lines (HaCaT, HEK293, and RAW264.7 cells) were purchased from American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM), RPMI1640, and penicillin-streptomycin were purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) and phosphate buffer saline (PBS) were obtained from Biotechnics Research (Lake Forest, CA, USA) and Capricorn Scientific (Ebsdorfergrund, Germany), respectively. 3-(4-5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Solon, OH, USA). Polyethylenimine (PEI), 1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), ascorbic acid, sodium nitroprusside (SNP), dehydrorhodamine 123 (DHR123), and Bay11-7082 were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). The luciferase assay system was purchased from Promega (Madison, WI, USA). Polyvinylidenedifluoride (PVDF) membrane was purchased from Merck Millipore (Billerica, MA, USA). Antibodies against cleaved or total forms of caspase-3, caspase-8, and caspase-9, and phospho- or total forms of PI3K, PDK1, AKT, p65, p50, c-Jun, c-Fos, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA).

Han S.Y., Kim E., Hwang K., Ratan Z.A., Hwang H., Kim E.M., Kim D., Park J, & Cho J.Y. (2018). Cytoprotective Effect of Epigallocatechin Gallate (EGCG)-5′-O-α-Glucopyranoside, a Novel EGCG Derivative. International Journal of Molecular Sciences, 19(5), 1466.

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Western Blot Analysis of Inflammatory Signaling

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RAW264.7 cells and colon tissues were homogenized, and protein levels were quantified using BCA reagent (Beyotime, Shanghai, China). Nuclear and cytoplasmic extractions were performed as instructed by the manufacturer (Beyotime, Shanghai, China). Samples were loaded and electrophoresed in 10–12% SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk diluted in TBS-T for 2 h followed by incubation with primary antibodies. The following primary antibodies were used in this experiment: iNOS, COX-2, phospho-IKKα/β, IKKα/β, phospho-IκBα, IκBα, phospho-ERK ½, ERK ½, phospho-p38, p38, p65, phospho-c-Jun, c-Jun, c-Fos, β-tubulin, Lamin A/C, phospho-JNK, JNK, IRAK4, and β-actin at 1:1,000 dilution (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG at 1:2,500 dilution.

Lertnimitphun P., Jiang Y., Kim N., Fu W., Zheng C., Tan H., Zhou H., Zhang X., Pei W., Lu Y, & Xu H. (2019). Safranal Alleviates Dextran Sulfate Sodium-Induced Colitis and Suppresses Macrophage-Mediated Inflammation. Frontiers in Pharmacology, 10, 1281.

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LUAD Tissue and Cell Line Protocol

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Fresh resected human LUAD and matched adjacent noncancerous tissue were obtained after informed consent and approval by the institutional review board (16-1514, 16-107, and 12-245) at Memorial Sloan Kettering Cancer Center. Human LUAD cell lines, human lung fibroblast cells and a normal bronchial epithelial cell line (NL20) were obtained from American Type Culture Collection; Lenti-X 293T cells were purchased from Takara Bio. All cells were tested for mycoplasma. The primary antibodies used were BRMS1 (Abcam, ab134968), c-fos (Cell Signaling, #2250), c-Jun (Cell Signaling, #9165), V5-epitope (Abcam, ab15828), CEACAM6 (Santa Cruz, SC-59899), Src (Cell Signaling, #2109), p-Src Y416 (Cell Signaling, #59548), p-fos S32 (Cell Signaling, #5248), actin (Santa Cruz, sc-8432), p-Jun S63 (Cell Signaling, #2361), RAMP1 (Thermo Fisher, 10327-1-AP), and F5 (Thermo Fisher, PA5-103046). The reagents used—c-fos inhibitor T5224 (S8966) and Src inhibitor Saracatinib (AZD0530, S1006)—were purchased from Selleck Chemicals. Collagen Type IV (C6745), Collagen Type I solution (C3867), and TCN (T7760) were purchased from Millipore Sigma; puromycin (A1113803) and blasticidin (A1113903) were purchased from Thermo Fisher; CellTiter-Glo was obtained from Promega (G9241); and poly-HEMA solution was purchased from Sigma-Aldrich (P3932).

Liu Y., Chudgar N., Mastrogiacomo B., He D., Lankadasari M.B., Bapat S., Jones G.D., Sanchez-Vega F., Tan K.S., Schultz N., Mukherjee S., Offit K., Bao Y., Bott M.J., Rekhtman N., Adusumilli P.S., Li B.T., Mayo M.W, & Jones D.R. (2022). A germline SNP in BRMS1 predisposes patients with lung adenocarcinoma to metastasis and can be ameliorated by targeting c-fos. Science translational medicine, 14(665), eabo1050.

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Comprehensive Western Blot and Immunofluorescence Antibodies

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The following antibodies were used in Western blotting and immunofluorescence:
phospho-GSK3α/β (CST 8566), phospho-β-Catenin (Ser33/37/Thr41) (CST9561), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (CST 8814), total β-Catenin (CST 9587), phospho-cyclin D1 (CST 3300), PPARγ (CST 2443), p27 (CST 2552), Ki-67 (CST 9129), phospho-c-Jun (CST 9164), c-Myc (CST 13987), Cytochrome c (CST 4280),GFAP (CST 3670), vimentin (CST 5741), NF-kB (CST 8242), c-Fos (CST 2250) from Cell Signaling Technology (Danvers, MA, US); Lamin B1 (10H34L18), Cyclin D1 (MA5-14512), Bcl-2 (13-8800), phospho-CREB (MA1-083), Ki-67 (MA5-14520) from Invitrogen, Thermo Fisher Scientific, and Phospho-JNK (sc-6254) from Santa Cruz Biotechnology (Dallas, TX, USA).

Ratti S., Rusciano I., Mongiorgi S., Neri I., Cappellini A., Cortelli P., Suh P.G., McCubrey J.A., Manzoli L., Cocco L, & Ramazzotti G. (2021). Lamin B1 Accumulation’s Effects on Autosomal Dominant Leukodystrophy (ADLD): Induction of Reactivity in the Astrocytes. Cells, 10(10), 2566.

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Protein Extraction and Western Blot Analysis

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Skin was collected from the Acetone and TPA treated wild-type and K14-sPLA₂-IIA mice and flash-frozen in liquid Nitrogen. The epidermis was scraped and minced in the RIPA buffer containing protease and phosphatase inhibitors cocktail (Roche). The tissue was homogenized for 10 min and frozen at −80 °C until further use. For the preparation of the whole cell lysates of cultured cells, the cells were washed with ice-cold PBS and scraped in the RIPA buffer. Next, the cell lysate was centrifuged at 16,000 ×g for 30 min at 4 °C. Protein concentration was quantified using Bradford reagent (Sigma) as per manufacturer's instruction. Total 40–60 μg of protein was loaded on 10% SDS-polyacrylamide gel and transferred on to nitrocellulose membrane. Membrane was then blocked with blocking buffer (5% nonfat dry milk or 5% BSA, 10 mM Tris, 150 mM NaCl, 0.1% Tween-20) and probed with the following primary antibodies: p-c-Jun (1:1000), c-Jun (1:1000), p-c-Fos (1:1000), c-Fos (1:1000) all from Cell Signalling Technologies, USA, anti-sPLA₂-IIA (1:500) from Merck-Millipore and β-actin (1:10000) from Sigma Aldrich.

Chovatiya G.L., Sunkara R.R., Roy S., Godbole S.R, & Waghmare S.K. (2019). Context-dependent effect of sPLA2-IIA induced proliferation on murine hair follicle stem cells and human epithelial cancer. EBioMedicine, 48, 364-376.

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Immunohistochemical Analysis of Neuronal Markers

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All animals were perfused and collected brain tissues as same as mentioned above. Brain sections at 30 μm thickness were sliced for immunostaining studies, primary antibodies against ctGluR1(Rabbit, Invitrogen, PA5-99527), c-Fos (rabbit, Cell Signaling Technology, #2250), NR1(rabbit, ABclonal, A7677), VMAT2 (rabbit, Phoenix Pharmaceuticals, H-V008), or Mc4R (rabbit, Alomone labs, Cat#AMR-024). All brain sections were incubated with the primary antibodies overnight at room temperature following 1 h blocking in 10% normal donkey serum. After visualized with secondary donkey IgG serum conjugated with Alexa Fluor 488 or Alexa Fluor 647 (Jackson ImmunoResearch, West Grove, PA, USA), sections were photographed with a TCS SP5 confocal microscopy.

Xu Y., Jiang Z., Li H., Cai J., Jiang Y., Ortiz-Guzman J., Xu Y., Arenkiel B.R, & Tong Q. (2023). Lateral septum as a melanocortin downstream site in obesity development. Cell reports, 42(5), 112502.

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