Anti phospho crebser133 antibody

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 CREB^Ser133 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.

Chromatin immunoprecipitation (ChIP) analysis of 10T1/2 cells differentiated into brown-like adipocytes was performed according to the manufacturer’s protocol (Millipore, catalog no. 17-295), as previously described [21 (link)]. Briefly, differentiated cells were treated with 1% formaldehyde to cross-link protein to DNA. Cells were then washed with ice-cold PBS buffer containing protease inhibitors (Millipore, catalog no. 20-283), harvested, and sonicated for 30 minutes using Diagenode Bioruptor^®. Protein/DNA complexes were immunoprecipitated overnight at 4°C using anti–phospho-CREB Ser133 antibody (Cell Signaling Technology, catalog no. 9198S) [24 ] or with IgG control antibody (Millipore, catalog no. 17-295). After incubation with protein A agarose beads (Millipore, catalog no. 16-201C), protein/DNA complexes were eluted for 15 minutes at room temperature using Elution Reagent C (Millipore, catalog no. 20-294). DNA fragments were purified and amplified by qPCR using the following primers designed specifically to recognize the area spanning the putative CRE elements identified in the proximal ZNF638 promoter in position −463 to −456 and −416 to −409: PromZNF638, forward, 5′-CTCAGTGGTTAGGAGCACT-3′; PromoZNF638, reverse, 5′-GGCACCCCA GATTAGGAAT-3′.