Total protein extracts (30 µg) were then electrophoresed in SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were blocked in tris-buffered saline (TBS) (50mM Tris-HCl, pH 7.5, 150mM NaCl and 0.05% Tween 20) with 5% dry milk and incubated with the following primary antibodies overnight at 4°C: anti-Arc/Arg3.1 antibody (1:800; Santa Cruz Biotechnology), anti-phospho-p44/42 ERK1/2 antibody (1:2000; Cell Signaling), anti-ERK1/2 antibody (1:1000; Cell Signaling), anti-phospho CREBSer133 antibody (1:1000; Cell Signaling), anti-CREB antibody (1:1000; Cell Signaling), anti-GluR1/2/3 antibody (1:1000; Millipore), and anti-β-Actin antibody (1:5000; Sigma). After washing three times for 5min in TBS/0.1% Tween-20, blots were then incubated with anti-rabbit or -mouse secondary antibody conjugated to horseradish peroxidase (1:2000; Zhongshan Biotechnology) and developed using the West Dura chemiluminescent substrate (Pierce Laboratories). Densitometry was determined based on band intensity, and relative protein expression was quantified by densitometry using the Total Lab 2.01 analysis system (Phoretix). To control for inconsistencies in loading, optical densities were normalized to β-actin protein expression. Data for treated animals were normalized to the average value of the naive controls.
Anti creb antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-CREB antibody is a research-use only product that recognizes the CREB (cAMP-response element binding protein) transcription factor. CREB is a key regulator of gene expression involved in various cellular processes. This antibody can be used to detect and analyze CREB in biological samples through techniques such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti arc arg3.1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti phospho crebser133 antibody, by Cell Signaling Technology (1 mentions)
Anti nnos antibody, by Cell Signaling Technology (1 mentions)
Phospho creb ser133 antibody, by Cell Signaling Technology (1 mentions)
Anti pka antibody, by Cell Signaling Technology (1 mentions)
4 chloro dl phenylalanine pcpa, by Merck Group (1 mentions)
Anti sox10 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti phospho creb antibody, by Cell Signaling Technology (1 mentions)
Ta 09, by ZSGB-BIO (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
P creb ser133, by Cell Signaling Technology (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Ampkα1, by Cell Signaling Technology (1 mentions)
P ampk thr172, by Cell Signaling Technology (1 mentions)
Cat 2, by Santa Cruz Biotechnology (1 mentions)
Anti p erk, by Abcepta (1 mentions)
Anti ampk β, by Abcepta (1 mentions)
14 protocols using anti creb antibody
1
Western Blot Analysis of Synaptic Proteins
2
Anti-5-HT1AR Pathway Regulation in Kadsura
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fruits of Kadsura longipedunculata were purchased from Shenyang, Liaoling Province, People’s Republic of China, in May 2013 and were identified by Professor Jimin Xu (National Institutes for Food and Drug Control). Then, the voucher specimen (No. 20130511) was deposited in the Research Department of Natural Medicine, Shenyang Pharmaceutical University. Anti-5-HT1AR antibody (catalog no. 85615, Abcam, UK), anti-nNOS antibody (catalog no. 4231, Cell Signaling Technology, USA), anti-CREB antibody (catalog no. 9197, Cell Signaling Technology, USA), phospho-CREB (Ser133) antibody (catalog no. 9198, Cell Signaling Technology, USA), anti-PKA antibody (catalog no. 5842, Cell Signaling Technology, USA), phospho-PKA (Thr197) antibody (catalog no. 5661, Cell Signaling Technology, USA), 4-chloro-DL-phenylalanine (PCPA, catalog no. C6506, Sigma, USA) were acquired. An NO kit was obtained from Nanjing Jiancheng Bioengineering Institute (catalog no. 20141017, Jiangshu, China).
3
ChIP-PCR of CREB and SOX10
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were incubated with 1% formaldehyde (Sigma-Aldrich, F8775) to cross-link between DNA and proteins, and sonicated to yield chromatin fragments with about 200-500 base pairs. Chromatin fragments were reacted with anti-CREB antibody (Cell Signaling, 9197) or anti-SOX10 antibody (Santa Cruz, sc-17342) at 4℃ overnight, and precipitated with protein A-sepharose bead-sheared salmon sperm slurry (Merck, 17-295) for 4 h. Input and precipitated DNAs were subjected to PCR encompassing CREB- or SOX10-responsive element at the MITF-M promoter. Nucleotide sequences of PCR primers are previously described 35 (link). PCR products were resolved on agarose gels by electrophoresis and stained with EcoDye.
4
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The dissection and homogenization of the TG and Western blot analysis were performed as described in detail previously [32 (link)]. The primary antibodies were as follows: anti-Nav1.7 antibody (1:1000, 20257-1-AP, Proteintech, USA), anti-COX-2 antibody (1:1000, 12282s, Cell Signaling Technology, USA), anti-phospho-CREB antibody (1:1000, 9198s, Cell Signaling Technology, USA), anti-CREB antibody (1:1000, 9191s, Cell Signaling Technology, USA), anti-IL-1β antibody (1:500, YR0913021, R&D systems, USA), anti-glial fibrillary acidic protein (GFAP; a marker of glial activation) antibody (1:1000, P14136, Cell Signaling Technology, USA), and anti-β-actin antibody (1:10000, TA-09, ZSGB-BIO, China). The densities of the bands were quantified using the NIH ImageJ 1.38 software (NIH, Bethesda, MD, USA) and expressed as fold change of the control group after normalization to β-actin or CREB.
5
Immunoblotting and Flow Cytometry Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
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).
6
Chromatin Immunoprecipitation for Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following cell culture and fixation of samples, cross-linked chromatin were sheared by Covaris sonication system (S220) to desired fragment lengths of 300–500 base pairs. The sonicated products were incubated with Magnetic Dynabeads Protein G (1004, Life Tech) and linked with anti-KLF5 (ab137676, AbCam), anti-CREB antibody (#4820, Cell signaling), or Normal anti-IgG antibody (#2729, Cell signaling). ChIP-qPCR was performed using primers binding to the putative binding sites of MLK4 and PCK1, in order to calculate the relative enrichment of amplicons between the target antibody and anti-IgG antibody.
7
ChIP Assay of HUVECs for CREB Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay of HUVECs was performed using the Simple ChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology). Briefly, HUVECs were cross-linked with formaldehyde. DNA was treated by sonication and incubated with 1 μg rabbit IgG or anti-CREB antibody (Cell Signaling Technology). Immunoprecipitation was performed with the magnetic beads, and 2 μL immunoprecipitated DNA underwent PCR with the following primers: 5′-CTCTCCACGCCCTCAGGTAA-3′ and 5′-CGTCGGATAAGCAGTCGGAA-3′.
8
CREB Binding to DNA Repair Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA‐binding reactions were performed in a volume of 20 μL. Nuclear extract (10 μg) was incubated for 60 min at 4°C in binding buffer (100 mM Tris–HCl, 500 mM KCl, 10 mM DTT, 2.25% glycerol, 5 mM MgCl2, 0.05% NP‐40, 50 ng/μL poly‐dI‐dC, 0.05 mg/mL salmon sperm DNA, 10 μg/mL BSA). In super shift reactions, anti‐CREB antibody (Cell signaling, #9197) was added (0.3 μL). Then, 40 fmol 5′‐radiolabeled double‐stranded oligonucleotides were added, and incubation was continued at room temperature (RT) for 20 min. Oligonucleotides corresponded to in silico predicted CRE sites and immediate up‐ and downstream flanking promoter regions of selected BER genes (Table S3 ). In competitive reactions, 50‐fold excess of unlabeled oligonucleotide corresponding to the promoter region of POLB containing a previously established CRE site was added to the reaction mixture. Subsequently, glycerol was added to a final concentration of 5%, and samples were resolved on a 5% native polyacrylamide gel in 0.5× TBE buffer (70 V for 5 h at 4°C).
9
Chromatin Immunoprecipitation of MeCP2 and CREB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hippocampal tissue was dissected out and lysed by brief sonication in lysis buffer with a protease inhibitor. The clarified lysate (500 µg protein) was incubated with non-specific IgG (2 µg), polyclonal rabbit anti-MeCP2 antibody (2 µg, Cell Signaling) or anti-CREB antibody (2 µg, Cell Signaling) overnight at 4 °C. The protein A or G magnetic beads (GE Healthcare, Barrington, IL) were added to the IP reaction product in order to catch the immune complex at 4 °C for 3 h. The Immunoprecipitated complexes on the beads were washed three times with washing buffer. Immunoprecipitated samples or IgG control samples were washed with ice-cold lysis buffer and dissociated by heating for 5 min in the loading buffer, before being subjected to Western blot analysis using anti-CREB and anti-MeCP2 antibody, respectively.
10
Forskolin-induced CREB Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded onto 6 cm dishes at 1.0 × 105 cell/cm2. After 16 hr, the medium was replaced with DMEM supplemented with 1% (v/v) FBS, and the cells were then treated with 10 µM forskolin for 0–24 hr, respectively. Immunoblot analysis were performed as described previously73 (link). The first antibody (anti-phospho CREB (Ser133) antibody (Merck), anti-CREB antibody (Cell Signaling Technology, Danvers, MA, USA), anti-Nur77 antibody (Abcam, Cambridge, UK), anti-Synapsin1 antibody (Abcam), anti-NeuroD antibody (Cell Signaling Technology), anti-β-tubulin III antibody (Sigma) or anti-GAPDH antibody (Merck)) was loaded onto the membrane after blocking with 5% skimmed-milk (Nakarai, Kyoto, Japan), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (MBL CO., LTD., Nagoya, Japan). The bands were detected by ECL Select Western Blotting Detection System Reagent or ECL Prime Western Blotting Detection System Reagent and visualized with an Image Quant LAS 4000 (all GE Healthcare Life Sciences, Little Chalfont, UK). All measurements were made in triplicate. Quantitative analysis was performed by determining the immunofluorescence intensity of the target protein(s) using ImageJ. Data are presented as the mean ± SEM (n = 3). Statistical significance for each time was determined by Student’s t-test. *p < 0.05.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!