Dulbecco's modified Eagle's medium (DMEM) and other culture reagents were obtained from Gibco Life Technologies (Grand Island, NY).The cell culture plates were purchased from Nalge Nunc International (Roskilde, Denmark). Human insulin (HumulinR) was from Eli Lilly S.A.S.(Fegersheim, France). Bovine serum albumin (BSA), forskolin, IBMX, and dexamethasone were purchased from Sigma (St Louis, MO, USA). Compound C was purchased from Calbiochem (San Diego, CA). Anti-CREB, anti-phospho-CREB (Ser133), anti-PPARγ, anti-C/EBPα, anti-fatty acid synthase (FAS), anti-fatty acid binding protein 4 (FABP4), anti-C/EBPβ, anti-AMPK, anti-phospho-AMPK (Thr172), anti-acetyl-CoA carboxylase (ACC), anti-phospho-ACC(Ser79), anti-β-actin, anti-α1-tubulin, anti-mouse IgG and anti-rabbit IgG conjugated with horseradish peroxidase were from Cell Signaling Technology (Beverly, MA, USA). Murine-derived 3T3-L1 preadipocytes were purchased from American Type Culture Collection (Rockville, MD). Berberine was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
Anti phospho creb ser133
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-CREB (Ser133) is a laboratory reagent used to detect the phosphorylation of the transcription factor CREB at serine 133. It is a specific antibody that binds to the phosphorylated form of CREB, allowing for the identification and quantification of this post-translational modification.
Automatically generated - may contain errors
Lab products found in correlation
Anti creb, by Cell Signaling Technology (10 mentions)
Forskolin, by Merck Group (3 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti cyclin d1, by Cell Signaling Technology (2 mentions)
Anti gsα, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Anti phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Cell culture plates, by Thermo Fisher Scientific (1 mentions)
Human insulin humulinr, by Eli Lilly (1 mentions)
Anti fatty acid synthase, by Cell Signaling Technology (1 mentions)
Anti phospho ampk thr172, by Cell Signaling Technology (1 mentions)
Anti acetyl coa carboxylase, by Cell Signaling Technology (1 mentions)
Anti phospho acc ser79, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
19 protocols using anti phospho creb ser133
1
Berberine's Effects on Adipocyte Differentiation
2
Comprehensive Western Blotting Analysis
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Western Blotting analysis was performed as described previously 32 (link). Total cell extracts were lysed in cell lysis buffer (Beyotime, P0013), and the lysates were centrifuged at 12,000 rpm for 10 min at 4°C. Samples containing 30 µg protein were loaded on SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk or BSA and probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Blots were developed with lumi-light Western blotting substrate detection reagents (Roche, 12015200001). Nuclear- and cytoplasmic-enriched fractions were prepared using Nuclear-Cytosol Extraction Kit (Applygene, P1200), according to manufacturer's instructions. Western Blotting analysis was performed using the anti-p27 (Cell Signaling Technology, 3698S), anti-CREB (Cell Signaling Technology, 9197L), anti-phospho-CREB (Ser133) (Cell Signaling Technology, 9198L), anti-S-phase kinase-associated protein 2 (Skp2) (Cell Signaling Technology, 4313S), anti-CDK2 (Cell Signaling Technology, 2546S), anti-Cyclin E1 (Cell Signaling Technology, 4129S), PARP (Cell Signaling Technology, 9532S), anti-GAPDH (Cell Signaling Technology, 2118L), anti-β-arrestin 1 (Abcam, ab32099), anti-β-arrestin 2 (Abcam, ab54790), anti-tubulin (Beyotime, AT819), anti-cyclin D3 (Beyotime, AC856), anti-p21 (Beyotime, AP021).
3
Signaling Pathways in Cell Culture
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Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). LY294002, SB415286, DCFH-DA and MPP+ were purchased from Sigma Chemicals (St. Louis, MO, USA). PD98059, Anti-phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204), anti-ERK, anti-phospho-GSK3 β (Ser9), anti-GSK3β, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38 and anti-phospho-CREB (Ser133) were obtained from Cell Signaling Technology (Boston, MA, USA). The antibody against GAPDH and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
4
Phosphorylated CREB Stimulation Assay
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Cells were exposed to 10 μM SKF 38393 for 24 h to study phosphorylated cAMP binding protein (p-CREB) stimulation. Total protein was denatured and separated by electrophoresis on a 10% SDS–polyacrylamide gel and transferred to a PVDF membrane as described previously (Zhao et al., 2006 (link)). Membranes were incubated separately with primary anti-actin (Cedarlane, CLT9001) and anti-Phospho-CREB (Ser133, Cell Signaling Technology, 9198S) antibodies overnight at 4°C and then incubated separately with goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), and donkey anti-rabbit IgG antibody (GE Health Care, NA934VS). Blots were visualized using the Amersham ECL Prime Western Blotting Detection System (GE Health Care, RPN2232). Images were obtained using a Fusion FX5 and analyzed with Image J.
5
Endothelial Cell Culture and Signaling
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HEPES, human endothelial serum-free medium (HE-SFM), Fast DiI, Phalloidin-Alexa Fluor 546, Trizol Reagent, Superscript II, MMLV reverse transcriptase and protease inhibitor cocktail were from Life Technologies (Carlsbad, CA, USA); Medium 199 (M199), foetal bovine serum (FBS), heparin, forskolin, LPS, Tricaine, phytohemoagglutinin (PHA) and 1-phenyl-2-thiourea were from Sigma-Aldrich (Saint. Louis, MO, USA); L-glutamine, trypsin-EDTA, penicillin-streptomycin, were from Euroclone (Siziano, IT). Collagenase was from Worthington Biochemical Corporation (Lakewood, NJ, USA). Fibronectin was from Roche (Basilea, Switzerland). Matrigel was from Becton, Dickinson & Company (Frankin Lakes, NJ, USA). Goat anti-human IL-8 blocking antibody was from Abcam (Cambridge, UK); human recombinant VEGF-A was from Immunological Sciences (Rome, IT). Monoclonal antibody anti anti-actin (clone C4), KG-501 and QNZ were from Millipore (Billerica, MA, USA); rabbit polyclonal antibodies anti-phospho-CREB (ser133) and anti-phospho-p65 were from Cell Signalling Technology (Danvers, MA, USA).
6
MEDICA Synthesis and Signaling Pathway Analysis
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MEDICA [α,α′-tetramethyl hexadecanedioic acid, HOOC-C(CH3)2 -(CH2)12-C(CH3)2-COOH] was synthesized as previously described [21 (link)]. Anti-p21/WAF1 (#2946), anti-Cyclin D1(#2922), anti-phospho-RB(#9308), anti-β-Actin (#4967), anti-PARP (#9542), anti-cleaved Caspase3 (#9661), anti-phospho-Akt(Ser473) (#4060), anti-Akt (#9272), anti-phospho-STAT3(Tyr705) (#9145), anti-phospho-EGFR(Thr669) (#3056), anti-phospho-LKB1(Ser428) (#3051), anti-LKB1(#3050), anti-phospho-CREB(Ser133) (#9191), anti-CREB (#9197), anti-phospho-IRS-1(Ser636/639) (#2388), anti-phospho-ACC(Ser79)(#3661) antibodies were from Cell Signaling. Anti-EGFR(sc-03), anti-phospho-Erk(Tyr204) (sc-7383), anti-Erk (sc-93), anti-Met (sc-10), anti-STAT3 (sc-8019), anti-phospho-RSK1/2(Thr359/Ser363) (sc-12898), anti-RSK(sc-231), anti-phospho-STAT3(Ser727) (sc-8001), anti-IRS-1(sc-559) antibodies were from Santa Cruz Biotechnology. Anti-phospho-EGFR(Tyr1068) (ab40815), anti-phospho-HER(Tyr1248) (ab47755) antibodies were from Abcam. Anti-phospho-FAK(Ser910) (#445596) antibody was from Invitrogen. Anti-FAK(#61087) antibody was from BD. Anti-gp130 (#29731) antibody was from Upstate Biotechnology. Anti-α-Tubulin (T6074) antibody was from Sigma. EGF and HGF were from PeproTech. IL6 and IL6R were from R & D Systems. U0126 and PD0325901 were from Sigma.
7
Biochemical Analysis of Alzheimer's Pathways
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DL-2-amino-5-phosphonopentanoic acid (AP5), ifenprodil, 30% hydrogen peroxide (H2O2), piceid, forskolin, Hexafluoro-2-propanol (HFIP), glutaraldehyde, Trichloroacetic Acid (TCA) and human recombinant insulin were purchased from Sigma (St Louis, MO). Anti-MAP2 was from Sigma (St Louis, MO) Anti-APP (1G6) mouse monoclonal antibody raised against amino acid 573-596 of APP was purchased from Axxora; anti-APP (22C11) mouse monoclonal antibody against N terminus APP was developed in our laboratory; anti-Aβ (N terminus amino acid 1-12) mouse monoclonal antibody B436 was a gift from Dr. Steve L. Wagner (TorreyPines Therapeutics, Inc.); anti-Aβ (amino acid residues 17–24) mouse monoclonal antibody 4G8 was purchased from Covance. Anti-phospho-IGF-I Receptor β (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) (19H7), anti-IRβ, anti-phospho-CREB (Ser133), and anti-CREB antibodies were from Cell Signaling Technology (Danvers, MA).
8
Western Blotting of Endothelial Proteins
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Human umbilical vein endothelial cells (HUVECs) and organ tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors. Lysates were separated by SDS-PAGE and transferred to PVDF membranes. Target proteins were probed with specific antibodies overnight and then incubated with secondary antibodies conjugated with chemiluminescent molecules. This was followed by detection of chemiluminescent reagents (Millipore, Burlington, MA, United States) using the Bio-Rad ChemDoc MP system (Bio-Rad, Hercules, CA, United States). The primary antibodies were anti-Gsα (Santa Cruz, Dallas, TX, United States), anti-CREB (Cell Signaling Technology, Boston, MA, United States), anti-phospho-CREB Ser133 (Cell Signaling Technology), anti-GAPDH (Proteintech, Chicago, IL, United States), anti-PLVAP (Proteintech), anti-Na/K ATPase (Proteintech).
9
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).
10
Western Blot Analysis of Protein Expression
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The cells were lysed in RIPA lysis buffer with a protein inhibitor cocktail for 30 min on ice, and the lysates were centrifuged at 12,000×g for 10 min at 4°C. The supernatants were collected, and the protein concentrations were measured using a BCA protein assay kit (PP1002, Biocolor Bioscience and Technology Co., Shanghai, China). Thirty micrograms of each protein were separated by 12% or 15% SDS-PAGE before being transferred to polyvinylidene fluoride membranes (162-0177, Bio-Rad, Hercules, CA, USA). Western blotting was performed using the following primary antibodies: anti-GAPDH (97166, Cell Signaling Technology, Danvers, MA, USA), anti-CREB (9104S, Cell Signaling Technology), anti-phospho-CREB (Ser 133) (9196S, Cell Signaling Technology). The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology, 7074) and HRP-conjugated antimouse IgG antibodies (sc-2005, Santa Cruz Biotechnology, Dallas, TX, USA). After probing with specific primary antibodies and a HRP-conjugated secondary antibody, the protein bands were detected, and the band density was analyzed using ImageJ 1.41 (National Institutes of Health).
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