Western blotting procedures were performed as previously described (Son et al., 2014). The blots were incubated overnight at 4°C with rabbit anti-pCREB antibody (1:1,000; Cell Signaling Technology) and BDNF (1:500; Abcam, Cambridge, UK). After the membranes were extensively washed with PBS-T and incubated with horseradish peroxidase-conjugated anti-rabbit antibodies (1:10,000; Thermo Fisher Scientific, Rockford, IL, USA), signals were visualized using a chemiluminescence kit (SuperSignal West Pico; Thermo Fisher Scientific). The membranes were stripped and re-probed with an anti-β-actin antibody (1:20,000; Sigma-Aldrich) for normalization. The bands were quantified as optical density ratio of pCREB and BDNF to beta-actin using Scion Image Beta 4.0.2 for Windows XP software (Scion, Frederick, ME, USA).
Rabbit anti p creb antibody
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Rabbit anti-p-CREB antibody is a primary antibody that recognizes the phosphorylated form of the CREB (cAMP Response Element Binding) protein. CREB is a transcription factor that plays a key role in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti trkb antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti creb antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti β tubulin antibody, by Cell Signaling Technology (2 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti rabbit igg hrp linked antibody, by Wuhan Servicebio Technology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Rabbit anti gapdh antibody, by Abways (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Amitriptyline hydrochloride, by Merck Group (1 mentions)
Rabbit anti gapdh antibody, by Wuhan Servicebio Technology (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Bdnf antibody, by Abcam (1 mentions)
Lps escherichia coli 055 b5, by Merck Group (1 mentions)
5 protocols using rabbit anti p creb antibody
1
Analysis of CREB and BDNF Protein Levels
2
Western Blot Analysis of Protein Expression
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RIPA lysate buffer containing 1 mM PMSF was added to each sample to collect the total protein. The total protein concentration of each sample was determined by the BCA method and adjusted all samples to the same concentration. The protein samples were mixed with a 5x loading buffer and denatured at 95°C. The proteins were separated by SDS-PAGE (8% or 15%) and electrophoretically transferred onto polyvinylidene fluoride membranes. The membranes were probed with rabbit anti-TrkB antibody (1 : 1000; Cell Signaling Technology; Cat. No. #4603), rabbit anti-CREB antibody (1 : 1000; Cell Signaling Technology; Cat. No. #9197S), rabbit anti-p-CREB antibody (1 : 1000; Cell Signaling Technology; Cat. No. #9198S), rabbit anti-β-tubulin antibody (1 : 1000; Cell Signaling Technology; Cat. No. #2128), rabbit anti-synaptophysin polyclonal antibody (1:1000; Proteintech; Cat. No. #17785-1-AP), and rabbit anti-GAPDH antibody (1 : 1000; Servicebio; Cat. No. #GB11002) overnight at 4°C and then incubated with Anti-rabbit IgG HRP-Linked Antibody (1 : 3000; Servicebio; Cat. No. #GB23303) at 37°C for 1.5 h. Detection was performed using a ChemiDoc XRS+ (Bio-Rad, USA) image analysis system.
3
Protein Expression Analysis in Bone Tissues
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Cells were lysed in lysis buffer (RIPA buffer, 1 mM PMSF, Phosphatase inhibitor Cocktail and protease inhibitor cocktail) on ice for 15 min. Bone tissues were ground with a mortar in liquid nitrogen and were lysed in lysis buffer at 4 ˚C for 30 min. Protein fractions were collected by centrifugation at 12,000 × g, 4 ˚C for 10 min and then 10 mg of lysates were subjected to SDS-PAGE and transferred to nitrocellulose filter membrane (NC) membranes. The membranes were blocked with 5% skimmed milk and incubated with specific antibodies overnight. The antibody of used were listed: Rabbit anti-NFATc1 (1:1000, abclonal, A1539), rabbit anti-Ano1 (1:1000, abclonal, A10498), rabbit anti-p-CaMKIV antibody (1:1000, ImmunoWay, YP0043), rabbit anti-CaMKIV antibody (1:1000, Cell Signaling Technology, 4032), rabbit anti-Creb antibody (1:1000, Cell Signaling Technology, 9197), rabbit anti-p-Creb antibody (1:1000, Cell Signaling Technology, 9198), rabbit anti-Gapdh antibody (1:5000, Abways, AB0036).
4
Immunohistochemical Analysis of Hippocampal pCREB
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Control and POMCStarKO mice under random-fed conditions were anesthetized and transcardially perfused with saline followed by ice-cold 4% PFA (pH 7.4). The brains were dissected and post-fixed in 4% PFA at 4°C for 24 hours, and cryoprotected by a sequence of 10-30% sucrose in 1x PBS (pH 7.4) at 4°C until the tissue sunk to the bottom of the tube. Brains were cut at 30 μm-thickness on a freezing microtome and collected. Serial hippocampal sections were blocked with 2% chicken serum in KPBS + 0.4% Triton X-100 and 3% BSA and incubated with rabbit anti pCREB antibody (1:200; Cell signaling) in blocking solution overnight (16 hours) at 4°C. As secondary antibody, a chicken anti-rabbit Alexa Fluor 488 (1:250; Life Technologies) in KPBS + 0.4% Triton X-100 was used (2 hours at room temperature). Imaging was performed using a Leica DMI 6000B microscope with a 20x objective. Three mice per genotype were included for the analysis of the number of pCREB-positive cells in the dentate gyrus of the hippocampus. For each mouse, the mean number was calculated by averaging the number of pCREB-positive cells counted from four images.
5
Molecular Mechanisms of Antidepressant Effects
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Xiaoyao Pills (XYW) (TaiJi, China), fluoxetine hydrochloride (FLX) (Patheon, France) and Amitriptyline hydrochloride (Sigma, USA) were dissolved in 0.9% saline to prepare solutions. Paeoniflorin (C23H28O11), Liquiritin (C21H22O9), Glycyrrhizic acid (C42H62O16), Ligustilide (C12H14O2) (Cheng Du Ai Fa, China). LPS (Escherichia coli 055:B5) was provided by Sigma (St. Louis, MO). Rabbit anti-TrkB antibody (1:1000; Cell Signaling Technology; Cat. No. #4603), rabbit anti-cAMP response element-binding protein (CREB) antibody (1:1000; Cell Signaling Technology; Cat. No. #9197S), rabbit anti-p-CREB antibody (1:1000; Cell Signaling Technology; Cat. No. #9198S), rabbit anti-β-Tubulin antibody (1:1000; Cell Signaling Technology; Cat. No. #2128). Rabbit anti-brain-derived neurotrophic factor (BDNF) antibody (1:1000; Abcam; Cat. No. #ab108319). Rabbit anti-DLG4, PSD95-specific, polyclonal antibody (1:1000; Proteintech; Cat. No. #20665-1-AP) and rabbit anti-synaptophysin polyclonal antibody (1:1000; Proteintech; Cat. No. #17785-1-AP). Rabbit anti-GAPDH antibody (1:1000; Servicebio; Cat. No. #GB11002). Rabbit anti-IL-6 polyclonal antibody (1:300; Servicebio; Cat. No. #GB11117) and Alexa Fluor 488 goat anti-rabbit IgG (1:400; Servicebio; Cat. No. #GB25303).
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