Anti phospho c ebpβ thr235

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-phospho-C/EBPβ (Thr235) is a lab equipment product that detects phosphorylation of C/EBPβ (CCAAT/enhancer-binding protein beta) at threonine 235. It is used for research purposes.

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C/EBPβ (35 citations) Anti-C/EBPβ (13 citations)

4 protocols using anti phospho c ebpβ thr235

Analyzing Inflammasome Signaling in Neutrophils

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A total of 10⁶ neutrophils were lysed and analyzed by Western blotting as previously described (26 (link)) using the following antibodies: anti-IL-1β (3ZD; National Cancer Institute Biological Resources), anti-NLRP3 (D2P5E; Cell Signaling), anti-caspase-1 (Cell Signaling), anti-NF-κB p65 (D14E12; Cell Signaling), anti-phospho-NF-κB p65 (Ser536) (93H1; Cell Signaling), anti-IκBα (L35A5; Cell Signaling), anti-MyD88 (D80F5; Cell Signaling), anti-TRAF6 (D21G3; Cell Signaling), anti-IKKα (Cell Signaling), anti-IKKβ (2C8; Cell Signaling), anti-phospho-IKKα/β (Ser176/180) (16A6; Cell Signaling), anti-CREB1 (48H2; Cell Signaling), anti-phospho-CREB (Ser133) (87G3; Cell Signaling), anti-C/EBPβ (Cell Signaling), anti-phospho-C/EBPβ (Thr235) (Cell Signaling), or anti-β-actin (AC-15; Sigma-Aldrich). Peroxidase-conjugated secondary antibodies were used (BioLegend). Membranes were developed using ECL (Thermo Scientific) and detected using a Nikon camera as previously described (79 (link)). Quantification analysis of blots was performed using ImageJ, and β-actin was used as a loading control. The results for samples were expressed as a percentage of the value for the positive control (LPS or LPS+ATP group).

Lima T.S., Gov L, & Lodoen M.B. (2018). Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection. mBio, 9(1), e02027-17.

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Quantifying Protein Expression by Western Blot

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Western blot analysis was performed as previously described[19 ]. Briefly, cells were lysed in lysis buffer [150 mM NaCl, 1 % Triton X-100, 1% Sodium deoxycholate, 50 mM Tris-HCl (pH 7.5), 2 mM EDTA (pH 8.0), and 0.1% SDS]. 10∼50 μg of protein were separated on SDS PAGE, transferred to Nitrocellulose membrane, probed with anti-C/EBP-β or anti-phospho-C/EBP-β (Thr235)(1:1000, Cell Signaling) antibodies. Membranes were stripped and re-probed with anti β-actin antibody (1:1,000; Santa Cruz Biotechnology, CA, USA) to ensure equal loading. Densitometric analysis was performed using the imageJ 1.47v system.

Kim S., Song J.H., Kim S., Qu P., Martin B.K., Sehareen W.S., Haines D.C., Lin P.C., Sharan S.K, & Chang S. (2016). Loss of oncogenic miR-155 in tumor cells promotes tumor growth by enhancing C/EBP-β-mediated MDSC infiltration. Oncotarget, 7(10), 11094-11112.

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Transcription Factor Expression Analysis

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RNA extraction was processed according to the RNeasy (QIAGEN) protocol. After genomic DNA degradation with the RNase-Free DNase kit (QIAGEN), reverse transcription was performed with the Superscript III (Invitrogen). For Srebf1, Klf4, Klf5, Krox20, C/EBPδ, C/EBPβ, Prep1, Glut4 expression analysis, cDNAs were subjected to qRT-PCR on a Roche LightCycler480 (Roche) using predesigned primers (RealTime ready assays, Roche). Sequences are available upon request. Results were normalized to Gapdh gene expression.
Antibodies used were anti-Prep1 (Santa Cruz sc-25282), anti-C/EBPα (D56F10 Cell signaling Technology #8178), anti-C/EBPβ (sc-150, Santa Cruz Biotechnology, Santa Cruz, USA), anti-phospho-C/EBPβ (Thr235) (Cell Signaling Technology #3084), anti-C/EBPδ (sc-151, Santa Cruz Biotechnology, Santa Cruz, USA), anti-Pparγ (81B8, Cell signaling Technology #2443), anti-pIrs1 (Tyr941) (07-848-I Millipore), anti-pAkt (Ser473) (D9E Cell signaling Technology #4060), anti-pAkt (Thr308) (D25E6 Cell signaling Technology #13038), anti-Klf5 (Abcam ab24331), anti-Vinculin (Sigma V9131).

Maroni G., Tkachuk V.A., Egorov A., Morelli M.J., Luongo R., Levantini E., Blasi F., Magli M.C, & Penkov D. (2017). Prep1 prevents premature adipogenesis of mesenchymal progenitors. Scientific Reports, 7, 15573.

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Western Blot Analysis of Inflammatory Signaling

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Proteins in the lysate were separated on a denaturing polyacrylamide gel and transferred to a polyvinylidene fluoride (polyvinylidene difluoride) membrane (Merck Millipore, Burlington, MA). The membrane was incubated with 5% skim milk (BD Biosciences) in Tris-buffered saline with 0.1% Tween 20 (TBST) buffer and the primary antibodies, namely, anti-IL-1β (GTX130021; GeneTex, Irvine, CA), anti-GSDMDC1 (sc-81868; Santa Cruz Biotechnology), anti-β-actin (sc-47778; Santa Cruz Biotechnology), anti-caspase-1 (3866S; Cell Signaling Technology), anti-pan-flavivirus E (4G2, purified in the lab), anti-C3 (ab200999; Abcam), anti-C/EBP-β (90081S; Cell Signaling Technology), anti-C/EBP-β (LAP) (3087S; Cell Signaling Technology), anti-phospho-C/EBP-β (Thr235) (3084S; Cell Signaling Technology), anti-p38 MAPK (9212S; Cell Signaling Technology), anti-phospho-p38 MAPK (9211S; Cell Signaling Technology), anti-p44/42 MAPK (9102S; Cell Signaling Technology), and anti-phospho-p44/42 MAPK (4370S; Cell Signaling Technology). Horseradish peroxidase-conjugated secondary antibodies from Bio-Rad and enhanced chemiluminescence reagents (Thermo Fisher Scientific) were used for protein detection.

Jeong G.U., Lee S., Kim D.Y., Lyu J., Yoon G.Y., Kim K.D., Ku K.B., Ko J, & Kwon Y.C. (2023). Zika Virus Infection Induces Interleukin-1β-Mediated Inflammatory Responses by Macrophages in the Brain of an Adult Mouse Model. Journal of Virology, 97(6), e00556-23.

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