P creb ser133

Manufactured by Cell Signaling Technology

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P-CREB (Ser133) is a primary antibody product offered by Cell Signaling Technology. It is designed to detect phosphorylation of the transcription factor CREB at serine 133, a post-translational modification that regulates CREB activity.

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21 protocols using p creb ser133

Astrocyte Protein Extraction and Analysis

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The RIPA lysis buffer (Beyotime,
Shanghai, China) was used to extract proteins from the astrocytes
according to the instruction of the manufacturer, followed by protein
quantification using a BCA assay kit (Beyotime, Shanghai, China).
For each sample, 30 μg of total protein was loaded and separated
by 12% SDS-PAGE, followed by being transferred to the polyvinylidene
difluoride (PVDF) membrane. The membrane was subsequently incubated
with antibodies against iNOS (1:1000, Abcam, USA), COX-2 (1:1000,
Abcam, USA), p-CREB (Ser133) (1:1000, Cell Signaling Technologies,
USA), CREB (1:1000, Cell Signaling Technologies, USA), and BDNF (1:1000,
Abcam, USA), using β-actin as a negative control. The pictures
were taken and analyzed using Image J.

Zhang C., Xing Z., Tan M., Wu Y, & Zeng W. (2021). Roflumilast Ameliorates Isoflurane-Induced Inflammation in Astrocytes via the CREB/BDNF Signaling Pathway. ACS Omega, 6(6), 4167-4174.

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Comprehensive Western Blot Analysis

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Western blotting was performed, as previously described [20 (link)]. Western blot analysis was performed using primary antibodies against phosphorylated hormone-sensitive lipase (p-HSL Ser660, rabbit, Cell Signaling), hormone-sensitive lipase (HSL, rabbit, Cell Signaling), phosphorylated perilipin1 (p-PLIN1 Ser522, mouse, VALA science), perilipin1 (PLIN1, rabbit, Santacruz), beta-actin (mouse, Santacruz), phosphor-(Ser/Thr) protein kinase A substrates (p-PKA substrates, rabbit, Cell Signaling), sirtuin 1 (SIRT1, rabbit, Cell Signaling), Estrogen Receptor Alpha Antibody A300-498A (Estrogen receptor alpha (ERα), rabbit, Bethyl laboratories INC.), adipose triglyceride lipase (ATGL, rabbit, Cell Signaling), phosphor- cAMP-response element binding protein (p-CREB ser133, rabbit, Cell Signaling), cAMP-response element binding protein (CREB, rabbit, Cell Signaling), uncoupling protein 1 (UCP1, rabbit, Alpha Diagnostic International), total oxphos rodent WB antibody cocktail (mouse, Abcam: ATP synthase subunit alpha, mitochondrial (ATP5A), succinate dehydrogenase [ubiquinone] iron–sulfur subunit, mitochondrial (SDHB), NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial (NDUFB8), cytochrome b-c1 complex subunit 2, mitochondrial (UQCRC2)), and α/β tubulin (rabbit, Cell Signaling). Blots were visualized with Super Signal West Dura Substrate (Pierce, Invitrogen).

Kim M., Im S., Cho Y.K., Choi C., Son Y., Kwon D., Jung Y.S, & Lee Y.H. (2020). Anti-Obesity Effects of Soybean Embryo Extract and Enzymatically-Modified Isoquercitrin. Biomolecules, 10(10), 1394.

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Protein Expression Analysis by Western Blot

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Protein preparation and western blot analysis were performed as described previously (Hu et al., 2007 (link)). Briefly, samples were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, United States). The blots were blocked with non-fat dry milk (7.5%) in Tris-buffered saline/Tween 20 (TBST) for 2 h and then incubated with primary antibodies at 4°C overnight. Antibodies against p-CREB Ser133 (#9198), CREB (#9197), ERK1/2 (#4695), p-ERK1/2 Thr202/Tyr204 (#4370), p-STAT3 Tyr705 (#9145), STAT3 (#4904), and GAPDH (#5174) were purchased from Cell Signaling Technology (Danvers, MA, United States) and diluted at 1:1000 for use. The horseradish peroxidase-conjugated secondary antibodies (Dingguo, Beijing, China) were used at 1:20,000 dilutions for GAPDH and 1:5000 otherwise. Immunoreactive proteins were detected by ECL plus Western blotting detection reagent (GE Healthcare, Buckinghamshire, United Kingdom) and quantified by densitometry (Bio-Rad).

Xie W., Ye Y., Feng Y., Xu T., Huang S., Shen J, & Leng Y. (2018). Linderane Suppresses Hepatic Gluconeogenesis by Inhibiting the cAMP/PKA/CREB Pathway Through Indirect Activation of PDE 3 via ERK/STAT3. Frontiers in Pharmacology, 9, 476.

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Immunofluorescence Staining of ER Stress Markers

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Immunofluorescence (IF) staining was conducted as previously described (Naidoo et al., 2011 (link)). Primary antibodies are as follows: p‐CREB (ser133) (1:300, Cell Signaling 87G3); CREB (1:200, Cell Signaling 86B10); p‐PERK (Thr980) (1:200, Bioss bs‐3330R); ATF4 (1:500, ProteinTech, 60035‐1‐Ig); peIF2α (1:100, Cell Signaling 3597S); and BiP/anti‐KDEL (1:1000, Enzo Life Sciences ADI‐SPA‐827F). Secondary antibodies are as follows: Alexa Fluor 488 donkey anti‐rabbit IgG (1:500); Alex Flur 594 donkey anti‐mouse IgG (1:500); and Alexa Fluor 488 donkey anti‐mouse IgG (1:500).

Hafycz J.M., Strus E, & Naidoo N. (2022). Reducing ER stress with chaperone therapy reverses sleep fragmentation and cognitive decline in aged mice. Aging Cell, 21(6), e13598.

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Western Blot Analysis of Rat Hippocampus

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Following decapitation, rat hippocampus was isolated rapidly and stored at −80 °C for Western blotting detection. The tissues were weighed, sonicated in RIPA lysis buffer supplemented with fresh protease and phosphatase inhibitors, and centrifuged at a speed of 12,000 rpm for 25 min. Then the protein concentration was determined by BCA assay. After denaturation and electrophoresis, the proteins were transferred onto a PVDF membrane in wet conditions. Following blocking in Tris-buffered saline Tween-20 solution with 5% (w/v) nonfat milk powder (1 h, RT), the protein membranes were incubated respectively in primary antibody: rabbit anti-BDNF (1:400, Santa Cruz Biotechnology, Inc., CA, USA), cAMP response element binding protein (CREB, 1:1000, Cell Signaling Tech), p-CREB (Ser133, 1:400, Cell Signaling Tech, MA, USA) and β-actin (1:8000, Sigma–Aldrich Inc., MO, USA) at 4 °C overnight. After washing, the membranes were incubated (1 h, RT) with anti-rabbit or anti-mouse IgG labeled with horseradish peroxidase (Jackson ImmunoResearch Inc., PA, USA) and then the membranes were washed and developed with Chemiluminescence reagents (Millipore Corp., MA, USA). The chemiluminescence signal was transformed into a digital image using FUJIFILM imaging system LAS-3000 (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Ma H., Wang W., Xu S., Wang L, & Wang X. (2018). Potassium 2-(1-hydroxypentyl)-benzoate improves depressive-like behaviors in rat model. Acta Pharmaceutica Sinica. B, 8(6), 881-888.

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Immunofluorescence Staining of Neuronal Markers

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Coverslips were washed once with PBS and then fixed with 4% PFA at room temperature for 10 min and with methanol (prechilled in −20 °C) in −20 °C for 30 min. Cells were then permeabilized with 0.2% Triton-X-100 for 10 min. The cells were then blocked with blocking buffer (1% CCS, 4% BSA in PBS) for 1-2 h at room temperature. Primary antibodies, including Calnexin (Cell Signaling Technology, #2679P, 1:200), SV2A (SYSY, #119022, 1:500), SYNGR3 (santa cruz, # sc-271046, 1:200), SYNGR1 (SYSY, #103002, 1:1000), SYP (SYSY, #101011, 1:200), synapsin1 (synaptic systems, #106004, 1:500), PKA RIα (Cell Signal, #5675, 1:200), NeuN (Sigma-Aldrich, #MAB377, 1:1000), c-FOS (Abcam, #AB208942, 1:1000), c-FOS (SYSY, #226017, 1:1000), CREB (Abcam, #ab31387, 1:200), p-CREB Ser133 (Cell Signal, #9198S, 1:200), MAP2 (Abcam, #ab5392, 1:5000), p62 (MBL, #PM066, 1:1000), LC3B (Cell Signaling Technology, #3868S, 1:200), GFP (Abcam, #ab13970, 1:5000), diluted in blocking buffer containing 0.03% Triton-X-100 were incubated overnight at 4 °C. Secondary antibodies diluted in PBS containing 0.03% Triton-X-100 were incubated at room temperature for 1 h. Images were obtained using a confocal microscope with Zen 2011 software (Zeiss LSM 780, Carl Zeiss, Jena, Germany) at 40x and 63x and the figures were processed with Fiji (ImageJ) and Adobe Photoshop.

Zhou X., Lee Y.K., Li X., Kim H., Sanchez-Priego C., Han X., Tan H., Zhou S., Fu Y., Purtell K., Wang Q., Holstein G.R., Tang B., Peng J., Yang N, & Yue Z. (2024). Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons. Nature Communications, 15, 3113.

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Western Blot Analysis of γH2AX and pCREB

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Samples were homogenized in 100 µL of whole-cell lysis buffer (150 M NaCl, 0.5% dodecylmaltosid, 50 mMTris, pH 7.5, 0.5% Triton X-100) mixed with complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and then lysed on ice. Equal amounts of protein samples (100 µg) were subjected to 14% SDS-PAGE and transferred to polyvinylidenedifluoride membrane (Amersham, Freiburg, Germany). Primary antibodies for γH2AX (Ser139) (JWB 301, Millipore, Schwalbach, Germany) and pCREB (Ser133) (Cell Signaling, Frankfurt, Germany) were used at 1:1000 dilutions. Secondary peroxidase- conjugated antibodies (Chemicon, Temecula, CA, USA and Santa Cruz, Heidelberg, Germany) were used at 1:10000, and the ECL Western Blotting Substrate (Pierce Thermo Fisher Scientific, Darmstadt, Germany) was used for visualizing the antibody-bound protein. To confirm equal protein loading, membranes were subsequently re-probed with anti-GABDH or anti- β-actin antibodies (Santa Cruz Biotechnology, Heidelberg, Germany).

Müller-Längle A., Lutz H., Hehlgans S., Rödel F., Rau K, & Laube B. (2019). NMDA Receptor-Mediated Signaling Pathways Enhance Radiation Resistance, Survival and Migration in Glioblastoma Cells—A Potential Target for Adjuvant Radiotherapy. Cancers, 11(4), 503.

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Western Blot Antibody Validation

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Antibodies against phosphorylated-cAMP response element binding (CREB) protein (p-CREB; Ser133), and CREB (48H2) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA); against p-extracellular signal-regulated kinases (ERK) 1/2 (Thr202/Tyr204), ERK1/2 (MK1) and BDNF (N-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A monoclonal anti-β-actin antibody, methylthiazolyldiphenyl-tetrazolium bromide (MTT) and melatonin were provided by Sigma-Aldrich (St. Louis, MO, USA).

Heo J.C., Park J.A., Kim D.K, & Lee J.H. (2019). Photobiomodulation (660 nm) therapy reduces oxidative stress and induces BDNF expression in the hippocampus. Scientific Reports, 9, 10114.

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Phospho-proteomic analysis of hepatocytes

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IP was performed on hepatocyte (which were isolated from three animals and transduced as indicated in the specific figures) lysates using antibodies against LxRxx[S*/T*] and Rxx[S*/T*] phospho-motifs (both Cell Signaling Technology) with Pierce Protein A/G Magnetic Beads according to the manufacturer’s protocol. Briefly, 4 mg of protein (2 mg/ml) and 30 μg of antibody were used for each IP. Samples were eluted in 1× NuPAGE lithium dodecyl sulfate sample buffer supplemented with 60 mM dichlorodiphenyltrichloroethane (DDT) for 10 min at 95°C. Beads were magnetically separated from the immunoprecipitated product, which was further analyzed on WB or by Mass spectrometry. Tissues samples were lysed in radioimmunoprecipitation assay buffer (RIPA buffer) and blotted using classical WB techniques. Antibodies against p-PKD3 S744/748, PKD3/PKCν, pPKA Thr197, pCREB Ser133, Vinculin, and PKA Substrate Antibody were purchased from Cell Signaling Technology. GAPDH antibody was purchased from Thermo.

Loza-Valdes A., Mayer A.E., Kassouf T., Trujillo-Viera J., Schmitz W., Dziaczkowski F., Leitges M., Schlosser A, & Sumara G. (2021). A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism. Life Science Alliance, 4(8), e202000863.

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Immunoblotting and Flow Cytometry Protocols

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For immunoblotting, antibody against ubiquitin (Ub), AMPK‐α1, p‐AMPK (Thr172), Akt, p‐Akt (Thr308), p‐Akt (Ser473), ERK1/2, LC3‐I/II, or p‐CREB (Ser133) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐MAGE‐A3, anti‐ASS1, anti‐p‐ERK, anti‐AMPK‐β, anti‐AMPK‐r, anti‐TRIM28, and anti‐CREB antibodies were separately, obtained from Abgent (San Diego, CA, USA), BD Biosciences (San Jose, CA, USA), Polaris, Sigma‐Aldrich, and GeneTex (Irvine, CA, USA). Anti‐RNF44 antibody was purchased from Abcam (Cambridge, MA, USA). All secondary antibodies conjugated with HRP were purchased from Promega (Madison, WI, USA). The immunoblots were developed using ultrasensitive enhanced chemiluminescent substrate and visualized by ChemiDoc MP System (Bio‐Rad, Hercules, CA, USA). The anti‐CREB antibody used for chromatin immunoprecipitation (ChIP) was purchased from Cell Signaling Technology.
For CAT‐1 and CAT‐2 detection, melanoma cells were incubated with primary antibody (CAT‐1, Novus, Littleton, CO, USA, 1 : 20; CAT‐2, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 20) for 20 min and then incubated with second antibody conjugated with Alexa Fluor^® 555 (Thermo Fisher Scientific) for 20 min at room temperature. The samples were analyzed by FACS (BD Accuri™ C6).

Li Y., Wu C., Shah S.S., Chen S., Wangpaichitr M., Kuo M.T., Feun L.G., Han X., Suarez M., Prince J, & Savaraj N. (2017). Degradation of AMPK‐α1 sensitizes BRAF inhibitor‐resistant melanoma cells to arginine deprivation. Molecular Oncology, 11(12), 1806-1825.

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