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2 protocols using rabbit anti p akt 473

1

Rosemary Extract Neuroprotective Mechanisms

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Rosemary extracts (Kunming Pharmaceutical Co., China.) are the crucial active constituents extracted from Rosmarinus officinalis Linn, containing 60% carnosic acid. RE were dissolved in 1% Tween-80 to prepare a suspension with the concentration of 100 mg/mL. Rabbit anti-BDNF, rabbit anti-Iba1 and rabbit anti-IL-1β were purchased from Abcam (Shanghai, China). Rabbit anti-TNF-α, rabbit anti-p-NF κ B p65, rabbit anti-NF κ B p65, rabbit anti-AKT, rabbit anti-p-AKT 473, β-actin and horseradish peroxidase conjugated anti-rabbit IgG were acquired from Cell Signaling Technology (Boston, MA, United States). Enzyme-linked immunosorbent assay (ELISA) kits were obtained from RD, Bio-Techne (Emeryville, CA, United States). E.Z.N.A. stool DNA Kit was purchased from Omega Bio-tek (Norcross, GA, United States), AxyPrep DNA Gel Extraction Kit was acquired from Axygen Biosciences (Union City, CA, United States).
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2

Quantitative Analysis of Protein Abundance

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The concentrations of proteins in the islets and INS-1 cell lysates were quantitated by BCA protein assay (Pierce). Nuclear and cytoplasmic lysates were prepared with the NE-PER Kit (Pierce) according to the manufacturer's instructions. Protein samples (20 μg) were separated by electrophoresis with an 8% SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane, followed by immunoblotting according to the protocol outlined by Cell Signaling Technology. The blotted membrane was developed with ECL Advance (Cell Signaling) and imaged with a LAS-4000 Super CCD Remote Control Science Imaging System (GE). The relative abundance was quantified by densitometry using Quantity One 4.6.2 software (Bio-Rad). The following antibodies were used: mouse anti-SFRP5 (1:500, Santa Cruz Biotech), mouse anti-cyclin D2 (1:700, Abcam), rabbit anti-pAKT(473) (1:1000, Cell Signaling), rabbit anti-AKT (1:1000, Cell Signaling), mouse anti-tubulin (1:20,000, Sigma-Aldrich), and mouse anti-actin (1:1000, Santa Cruz).
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