Creb1

Manufactured by Cell Signaling Technology

Sourced in United States

CREB1 is a protein that functions as a transcription factor. It regulates the expression of genes involved in various cellular processes, including cell growth, differentiation, and survival.

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14 protocols using creb1

Western Blot Analysis of CREB, Nrf-1, and Ligase IV

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Western blotting assay was carried out as a standard protocol. The following antibodies were used: CREB1 (Cell Signaling Technology, Beverly, MA, USA), P-CREB1 (Cell Signaling Technology), Nrf-1 (Santa Cruz, CA, USA) and ligase IV (Santa Cruz), respectively. The membrane was incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody (Cell Signaling Technology). GAPDH (Proteintech, Chicago, USA) served as a loading control. Protein bands were detected using an Enhanced Chemiluminescence Detection System (Millipore, Billerica, MA, USA).

Yang Y., Wu N., Tian S., Li F., Hu H., Chen P., Cai X., Xu L., Zhang J., Chen Z., Ge J., Yu K, & Zhuang J. (2016). Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV. Cell Death & Disease, 7(11), e2473-.

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Liver Protein Expression Analysis

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The liver lysate samples of 30 μg per lane of gel were used for the SDS-PAGE and western blot. The used antibodies are listed below: G6PC (1:500), Sigma Aldrich, HPA052324-25UL; PCK1 (1:1000), Santa Cruz, sc-74825; LEPR (1:500), Novus Bio, NB120-5593; FOXO1 (1:1000), Cell Signaling, 2880 S; CREB1 (1:1000), Cell Signaling, 9197 S; GAPDH (1:4000), Cell Signaling, 2118 S.

Fang Z., Fan M., Yuan D., Jin L., Wang Y., Ding L., Xu S., Tu J., Zhang E., Wu X., Chen Z.B, & Huang W. (2023). Downregulation of hepatic lncRNA Gm19619 improves gluconeogenesis and lipogenesis following vertical sleeve gastrectomy in mice. Communications Biology, 6, 105.

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Investigating TGF-β Signaling Pathways

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Antibodies for E-cadherin, N-cadherin, vimentin, fibronectin, CREB1, pCREB1, pSMAD2/3, and CD44 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for TGFβ1, TGFβ2, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody for SRGN and anti-Rabbit FITC second antibody were purchased from Abcam (Cambridge, MA, USA). The recombinant human TGFβ1 and TGFβ2 protein was obtained from R&D Systems (Minneapolis, MN, USA). The TGFβ receptor I kinase inhibitor SD208 was from Selleckchem (Houston, TX, USA) and MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) was from Promega (Madison, WI, USA). siRNA of CD44 (siRCD44) was purchased from RiboBio (Guangzhou, China) and transformed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Zhang Z., Deng Y., Zheng G., Jia X., Xiong Y., Luo K., Qiu Q., Qiu N., Yin J., Lu M., Liu H., Gu Y, & He Z. (2017). SRGN-TGFβ2 regulatory loop confers invasion and metastasis in triple-negative breast cancer. Oncogenesis, 6(7), e360-.

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Western Blot Analysis of Signaling Proteins

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The cells were washed with PBS twice and lysed using lysis buffer on ice for 30 min. Protein fractions were collected by centrifugation at 14 000 r.p.m. at 4 °C for 15 min and then subjected to SDS–PAGE and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with 5% BSA and incubated overnight at 4 °C in primary antibody CREB5 (1:1000, Sigma, St Louis, MO, USA), CREB1 (1:1000, Cell Signaling Technology, Beverly, MA, USA), p–CREB (1:1000, Cell Signaling Technology), p65 (1:1000, Cell Signaling Technology), p–p65 (1:1000, Cell Signaling Technology) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:10 000, Bioworld Technology, St. Louis Park, MN, USA). The blots were washed three times with TBST buffer and then incubated for 1 h at room temperature with anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase. Western blot analysis was visualized using an enhanced chemiluminescence kit (Pierce, Beverly, MA, USA).

Long X., Li Y., Qiu S., Liu J., He L, & Peng Y. (2016). MiR-582-5p/miR-590-5p targeted CREB1/CREB5–NF-κB signaling and caused opioid-induced immunosuppression in human monocytes. Translational Psychiatry, 6(3), e757-.

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Gene Silencing and Luciferase Assay

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For gene silencing, cells were transfected at ∼60% confluence (60,000 cells/well in 12-well plates) in complete medium with 30 nM small interfering RNA (siRNA) targeting CREB1 (Cell Signaling Technology), ATF2 (Cell Signaling Technology), CREB3 (Thermo Fisher Scientific), or catalytic α subunit of PKA (PKACα; Cell Signaling Technology) using Lipofectamine RNAiMax Reagent (Thermo Fisher Scientific). For gene silencing followed by reporter assays, cells were first transduced with the RRAS-1907/+1-Fluc lentivirus and then transfected with siRNA in 96-well plates at a density of 15,000 cells/well. Luciferase activity was measured 72 h later using the Dual-Glo Luciferase Kit from Promega.

Perrot C.Y., Sawada J, & Komatsu M. (2018). Prolonged activation of cAMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS. The FASEB Journal, 32(11), 5793-5812.

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Standardized Herbal Decoction: Extraction and Quality Control

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MO, FA, RR and NR in SHD were purchased from the pharmacy of Xijing Hospital, and all were included in the Chinese Pharmacopoeia. The plant materials were authenticated by Professor Haifeng Tang. Voucher specimen (20C266, 20C315, 20C364, 20C375) was preserved in the herbarium of Xijing Hospital. SHD extract was prepared according to the following experimental steps: first, the essential oil was extracted, boiled in water and extracted three times. Then, the extracting solution was filtered and condensed by rotary evaporation. Finally, the solution was concentrated to 1 g/mL. The original solution was stored in a 4 °C refrigerator. The quality control of SHD is shown in Supplementary materials. The anti-PI3k, p-PI3k, Akt, p-Akt, CREB1, p-CREB1, TNF-α, IL-6 and β-actin for Western blotting were purchased from Cell Signaling Technology (Danvers, MA).

Zhang W., Zhang L., Wang W.J., Ma S., Wang M., Yao M., Li R., Li W.W., Zhao X., Hu D., Ding Y, & Wang J. (2022). Network pharmacology and in vitro experimental verification to explore the mechanism of Sanhua decoction in the treatment of ischaemic stroke. Pharmaceutical Biology, 60(1), 119-130.

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ATF4 and CREB1 Binding in MC3T3-E1 Cells

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MC3T3-E1 cells were expanded until confluent and treated with either 2.5 μM NE or vehicle control for 48 hours. DNA was subsequently extracted. All manufacturer instructions were subsequently followed for chromatin immunoprecipitation (CHiP) assay (Qiagen, cat. #334471). Antibodies were used for ATF4 (Cell Signaling, cat. #D4B8) and CREB1 (Cell Signaling, cat. #9197T). Primers were used for ATF4 (ThermoFisher, cat. #Mm00515325) and CREB1 (ThermoFisher cat. #Mm00501607). Data are presented as differences in promoter region amplification relative to IgG control and were statistically compared using two-way ANOVA with Sidak's multiple-comparisons test.

Decker A.M., Decker J.T., Jung Y., Cackowski F.C., Daignault-Newton S., Morgan T.M., Shea L.D, & Taichman R.S. (2020). Adrenergic Blockade Promotes Maintenance of Dormancy in Prostate Cancer Through Upregulation of GAS6. Translational Oncology, 13(7), 100781.

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Quercitrin Modulates Apoptosis Signaling

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DPCs (1 × 10 ⁶ cells/dish) were seeded in 100 mm dishes and cultured for 24 h. Quercitrin was treated at concentrations of 1, 10, and 100 nM for appropriate time. The cells were then lysed and total cellular proteins were prepared. 50 μg protein samples were analyzed by Western blotting with corresponding antibodies; Bcl2 (1:1000, Abcam, cambridge, UK), Akt (1:1000, Santa Cruz, CA, USA), Erk (p44/42) (1:1000, Cell Signaling Technology, Danvers, MA, USA), CREB-1 (Cell Signaling Technology, Danvers, MA, USA) GAPDH (1:2000, Santa Cruz, CA, USA), phospho (Ser473)-Akt (1:1000, Cell Signaling Technology, Danvers, MA, USA), phospho (Thr202/Tyr204)-Erk (p44/42) (1:1000, Cell Signaling Technology, Danvers, MA, USA), and phospho (Ser133)-CREB (1:1000, Cell Signaling Technology, Danvers, MA, USA). Western blot was analyzed by chemiluminescence detector iBright FL1000 (Invitrogen, Waltham, MA, USA).

Kim J., Kim S.R., Choi Y.H., Shin J.Y., Kim C.D., Kang N.G., Park B.C, & Lee S. (2020). Quercitrin Stimulates Hair Growth with Enhanced Expression of Growth Factors via Activation of MAPK/CREB Signaling Pathway. Molecules, 25(17), 4004.

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Immunoblot Analysis of Protein Expression

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Immunoblot analyses were performed using a range between 20 μg and 100 μg of lysates depending on the abundance of the protein of interest. The material was electrophoretically resolved on denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels ranging from 6 % to 16 % acrylamide and was transferred to nitrocellulose Immobilon-P membranes (Invitrogen) using the iBlot™ Dry Blotting System (Invitrogen). Membranes were then blocked in 5 % w/v Marvel™ milk or 5 % BSA (Sigma-Aldrich) w/v in 1× TBS-0.1 % Tween for 30 min at RT and were immunoblotted with β-Actin (Abcam), β-Tubulin (Abcam), Bak (Cell Signaling), Bax (Cell Signaling), Bid (Cell Signaling), CREB1 (Cell Signaling), pCREB1 (Cell Signaling), Cyclin D1 (Cell Signaling), Histone H3 (Abcam), p21 (Cell Signaling), p27 (Cell Signaling), PARP (Cell Signaling), SIK2 (BioLegend, Sigma-Aldrich or Cell Signaling), TORC1 (Cell Signaling), TORC2 (Cell Signaling) and TORC3 (Cell Signaling) primary antibodies overnight at 4°C. Membranes were then washed 5 times in 1× TBS-0.1 % Tween and were incubated with an appropriated HRP-conjugated secondary antibody (Dako) for 1 h at RT. Membranes were then washed 5 times in 1× TBS-0.1 % Tween and proteins were visualised using ECL Plus Western Blotting Detection System (GE Healthcare).

Bon H., Wadhwa K., Schreiner A., Osborne M., Carroll T., Ramos-Montoya A., Ross-Adams H., Visser M., Hoffmann R., Ahmed A.A., Neal D.E, & Mills I.G. (2014). Salt-inducible Kinase 2 Regulates Mitotic Progression and Transcription in Prostate Cancer. Molecular cancer research : MCR, 13(4), 620-635.

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Quantitative Western Blot Analysis

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After extraction and separation by SDS‐polyacrylamide gel electrophoresis in 10% Tris‐glycine gels, the protein samples were transferred to a nitrocellulose membrane. Primary antibodies including MeCP2 (Santa Cruz Biotechnology, Texas, USA) (dilution: 1:500), PSD95 (1:500) (ABclonal, Wuhan, China), CREB1 (Cell Signaling Technology, Inc., Danvers, USA) (dilution: 1:700), p‐CREB1 (ser133) (Cell Signaling Technology, Inc., Danvers, USA) (dilution: 1:700), and GAPDH (Proteintech Group, Inc, Wuhan China) (dilution: 1:10000) were incubated at 4℃ overnight. Subsequently, IRDye purified secondary antibody (dilution: 1:10000) was incubated. Immunopositive bands were visualized at Ex/Em=778nm/795nm.

Huang J., Zhang F., Su M., Li J., Yi W., Hou L., Yang S., Liu J., Zhang H., Ma T, & Wu D. (2021). MeCP2 prevents age‐associated cognitive decline via restoring synaptic plasticity in a senescence‐accelerated mouse model. Aging Cell, 20(9), e13451.

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