The antibodies used in this study were purchased from companies listed in Table 1 (primary antibodies) and Table 2 (secondary antibodies). Anti-pCREB was purchased from Cell Signaling (Danvers, MA) as either an unconjugated antibody (Anti-pCREB; catalog #9198) or as a conjugate to Alexa Fluor 488 (Anti-pCREB- Alexa Fluor 488 conjugate; catalog #9187; see Table 1 ). Two antibodies were directly conjugated to florescent dyes in our laboratory as follows. The antibody for SOX9 (#AB5535; Millipore; Billerica, MA) was conjugated to CF555 by diluting it 1:2 with the Mix-n-Stain CF555 antibody labeling kit (Biotium, Inc.; Hayward, CA) according to the manufacturer’s directions. Similarly, Iba1 (#019–19741; Wako Chemicals USA, Inc.; Richmond, VA) was conjugated to CF488A by diluting it 1:2 using the Mix-n-Stain CF488A kit from the same company. Hoechst33258 was purchased from ThermoFisher Scientific Inc. (Pittsburgh, PA). ProLong Gold antifade mounting media containing 4',6-diamidino-2-phenylindole (DAPI) was purchased from ThermoFisher Scientific Inc. λ-phosphatase was purchased from New England Biolabs (Ipswich, MA).
Anti pcreb
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
Anti-pCREB is a primary antibody that detects phosphorylated CREB (cAMP response element-binding protein) by immunoblotting and immunohistochemistry. CREB is a transcription factor that plays a key role in cellular processes such as memory formation, neuroplasticity, and cell survival.
Automatically generated - may contain errors
Lab products found in correlation
Anti creb, by Cell Signaling Technology (22 mentions)
Anti p akt, by Cell Signaling Technology (6 mentions)
Anti gapdh, by Cell Signaling Technology (5 mentions)
Anti p erk, by Cell Signaling Technology (5 mentions)
Anti p erk1 2, by Cell Signaling Technology (4 mentions)
Anti bdnf, by Abcam (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti camkii, by Cell Signaling Technology (3 mentions)
Anti erk, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
N 2 hexahydro 2 oxo 1h azepin 3 yl amino carbonyl phenyl benzo b thiophene 2 carboxamide ana 12, by Merck Group (2 mentions)
Anti trkb, by Cell Signaling Technology (2 mentions)
Anti α tubulin, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti foxo1, by Cell Signaling Technology (2 mentions)
Anti pparγ, by Cell Signaling Technology (2 mentions)
Anti pka c α, by Cell Signaling Technology (2 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti tcreb, by Cell Signaling Technology (2 mentions)
53 protocols using anti pcreb
1
Antibody Preparation and Characterization
2
Fermented Perilla frutescens Bioactive Compounds
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Fermented Perilla frutescens (FPF), uracil, adenine, PCA, L7dGn, A7dGn and L7Gn were provided by Huons Co Ltd. (Seoul, Republic of Korea). Corticosterone, FXT and N-[2-[(hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl-benzo[b]thiophene-2-carboxamide (ANA-12) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Corticosterone enzyme-linked immunosorbent assay (ELISA) kit and dopamine ELISA kit were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Serotonin ELISA kit and ACTH ELISA kit were purchased from Abcam (Cambridge, UK). Anti-BDNF, anti-CREB, anti-pCREB, anti-ERK, anti-pERK, anti-TrkB, and anti-GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-pTrkB antibody was purchased from Abcam (Cambridge, MA, USA). All other chemical reagents were purchased from Sigma-Aldrich and were of analytical or HPLC grade.
3
Western Blot Analysis of Signaling Proteins
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Cells were washed in ice-cold PBS and lysed in the lysis buffer (30 mM Tris, 200 mM NaCl, 1.5 mM MgCL2, 0.4 mM EDTA, 20% Glycerol, 1% NP-40, 1 mM DTT) with Complete Mini protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science). The protein concentration of cell lysates was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of cell lysates were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membranes were blocked with TBST (50mM Tris-HCI, pH8.0, 100mM NaCl and 0.1% Tween 20) containing 5% nonfat dried milk or BSA for 1 h at room temperature, and then probed with the indicated primary antibodies overnight at 4°C. The primary antibodies used were as follows: Anti-GRK3 (Epitomics, USA), Anti-CREB (Cell signaling, USA), Anti-p-CREB (Cell signaling, USA), Anti-Actin (Santa Cruz, USA). After washes, the membranes were incubated with HRP-conjugated secondary anti-mouse or anti-rabbit antibodies (Cell Signaling Technology) for 1 h at room temperature. Finally, the immunoreactive bands were developed with Pierce ECL Western Blotting Substrate (Thermo Scientific, USA) on Blue Basic autoradiography Film (Bioexpress).
4
Protein Expression Analysis in Hippocampus
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The protein samples (50 µg of protein) were prepared from micro-dissected hippocampus and separated by 12% SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes using a Bio-Rad Miniprotein III System wet transfer unit for 1 h at 4 °C. Transfer membranes were then incubated with blocking solution (5% nonfat dried milk dissolved in TBST buffer, pH 7.5) for 1 h at room temperature, then incubated with anti-BDNF (1:1000, Abcam), anti-PKA(1:1000, Imagenex), anti-CREB (1:1000, Cell Signaling), anti-pCREB (Ser133, 1:1000, Cell Signaling), anti-MeCP2 (1:2000, Cell Signaling), anti-pMeCP2 (Ser421, 1:1000, Abgent), anti-TrkB (1:1000, Millipore) and anti-pTrkB (Tyr706, 1:1000, Cell Signaling) overnight at 4 °C. Membranes were incubated with secondary antibodies for 1 hat 4 °C. Signal detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences) and quantitated by using the GS-710 Calibrated Image Densitometer (Bio-Rad).
5
Western Blot Analysis of Protein Expression
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Protein samples were diluted 1:5 with protein loading buffer (6 × Transgen Biotech, Beijing, China) and heated at 95 °C for 5 min. Protein extracts were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes, and blocked with 5% nonfat dry milk in TBST. The membranes were incubated with primary antibodies at 4 °C overnight and with the horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 1 hour. Protein bands were visualized by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA) and imaged by a FluorChem M gel documentation system (ProteinSimple, San Jose, CA). The primary antibodies (anti-YAP, anti-p-YAP, anti-LATS1, anti-p-LATS1, anti-MYPT1, anti-p-MYPT1, anti-CREB, anti-p-CREB, anti-GAPDH, anti-α-tubulin, anti-histone H3) and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). CTGF antibody was obtained from Sigma-Aldrich (St Louis, MO, USA).
6
Antibody Immunoblotting for Protein Analysis
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The following primary antibodies were used: anti-Akt (monoclonal rabbit, C67E7, #4691), anti-phospho (p)-Akt (S473, monoclonal rabbit, D9E, #4060), anti-Bcl-xL (monoclonal rabbit, 54H6, #2764), anti-Bcl-2 (polyclonal rabbit, #2876), anti-Cyclin D1 (monoclonal rabbit, 92G2, #2978), anti-CREB (monoclonal rabbit, 48H2, #9197), anti-p-CREB (S133, monoclonal rabbit, 87G3, #9198), anti-GAPDH (monoclonal rabbit, D16H11, #5174), anti-IGF-IR (monoclonal rabbit, D23H3, #9750 and monoclonal rabbit, D4O6W, #14,534), anti-phospho (p)-IGF-IR (Y1135/1136, monoclonal rabbit, 19H7, #3024), anti-PCNA (monoclonal rabbit, D3H8P, #13,110) and anti-Rb (polyclonal rabbit, #9313) (all purchased from Cell Signaling Technology). The SS18-SSX fusion protein was visualized using an anti-SS18/SYT antibody (#8819 Santa Cruz Biotechnology) detecting the N-terminus of SS18 (which is retained in the SS18-SSX fusion oncoprotein). Secondary antibody labeling (Bio-Rad) and immunoblot development were performed using an enhanced chemiluminescence detection kit (SignalFire ECL Reagent; Cell Signaling Technology) and a Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad), as described previously [14 (link), 40 (link)].
7
Immunohistochemical Analysis of Spinal Cord Proteins
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Under deep anesthesia with sodium pentobarbital at a dose of 60 mg kg−1, six male SD rats from each experimental group were transcardially perfused with 500 ml NS, followed by 500 ml 4% paraformaldehyde in PBS. The spinal cord was removed and post-fixed in 10% formalin solution for 12 h at 4°C. All samples were embedded in paraffin and transversely cut into 4-µm thick sections with a sliding microtome. Following deparaffinization and hydration, sections were blocked in 3% H2O2 for 10 min at room temperature, and were incubated with primary antibodies, including anti-NR2B (ab216621; 1:250; Abcam, Cambridge, UK), anti-p-CaMKIIα (ab5683; 1:300; Abcam), and anti-p-CREB (9198; 1:700; Cell Signaling Technology, Inc.), for 2 h at 37°C. Slides were washed with 0.1 M PBS 3 times for 2 min and subsequently incubated with a goat anti-rabbit secondary antibody (PV-9001; 1:500; ZSGB-BIO; OriGene Technologies, Inc., Rockville, MD, USA) for 30 min at 37°C, then stained with diaminobenzidine (DAB kit; ZSGB-BIO; OriGene Technologies, Inc.) and counterstained with hematoxylin for 30 sec at room temperature. Images were captured with a light microscope (Olympus Corporation, Tokyo, Japan). Quantitative image analysis of the relative optical density (OD) was performed using Image Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
8
Western Blot Analysis of ATF4 Pathway
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For Western immunoblotting analysis, treated neurons were collected in 1× LDS Loading Buffer (Thermo Fisher Scientific) and boiled for 10 min. Proteins were separated by electrophoresis in 10% NuPAGE gels (Thermo Fisher Scientific). To better detect ATF4, the gels were run a longer time (80–100 min) to separate ATF4 from a closely migrating nonspecific band. The following primary antibodies were used: rabbit monoclonal anti-ATF4 (1:1000), rabbit anti-eIF2a (1:1000), rabbit anti-p-eIF2a (1:1000), anti-CREB (1:1000), and anti-pCREB (1:2000), all from Cell Signaling Technology; and mouse anti-GAPDH (1:2000) from Imgenex. For secondary antibody, HRP-conjugated anti-rabbit and anti-mouse secondary antibody (1:5000; Thermo Fisher Scientific) was used. The ATF4 antibodies were validated by assessment in cultures in which ATF4 had been knocked down with lentiviral-delivered short hairpin ATF4 (shATF4; Corona et al., 2018 (link)).
9
Immunoblotting and Immunoprecipitation of Cavβ Subunits
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Cells were lysed in RIPA lysis buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA, and protease and phosphatase inhibitors. Cell lysate was incubated for 30 min at 4°C, followed by centrifugation at 16,900g for 15 min at 4°C. The protein concentration in the supernatant was determined, and Western blot and immunoprecipitation experiments were performed as described previously (19 (link)). Antibodies against the Cavβ1, β2, β3, and β4 were generated in-house (12 (link),17 (link),19 (link)). Other antibodies are as follows: anti-CREB (#4820) and anti–p-CREB (#9198) (Cell Signaling Technology); anti-MAFA (#NB400–137; Novus Biologicals); anti-IP3R type 3 (IP3R3 (#BD-610313; BD Biosciences); and anti-IP3R3 (#AB9076; Merck).
10
Chromatin Immunoprecipitation Assay for Transcription Factor Binding
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ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (9003; Cell Signaling Technology) according to the manufacturer’s protocol. Briefly, gastroc tissue specimens obtained from 8-week-old mice were minced into small pieces and cross-linked with 1% formaldehyde at room temperature for 10 minutes before quenching with a 0.125 M glycine solution. Tissue pieces were then resuspended in ice-cold phosphate-buffered saline (PBS) and ground with a Dounce homogenizer. The cross-linked chromatin fragments were then sheared using a sonicator (Scientz-IID; Scientz) into 200–1000 bp long fragments. Then, 10 μg of anti–p-CREB (9198; Cell Signaling Technology), anti–c-MYC (18583; Cell Signaling Technology), or control IgG (3900; Cell Signaling Technology) antibodies were used for immunoprecipitation. The protein-DNA cross-links were reversed overnight at 65°C, and the DNA fragments were purified and diluted for qPCR (details of the primers used are given in Supplemental Table 8 ).
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