Oligo primers

We treated RAW264.7 cells with 200 µg mL -1 GLPS for 0 and 24 h. Briefly, RNA was extracted from RAW264.7 by the Trizol method (Invitrogen, CA). To synthesize cDNA, total RNA was reverse transcribed with MMLV and oligo primers (Invitrogen, CA) in 20 μL reactions. A Bio-Rad system utilizing an SYBR Green I PCR Kit (Takara, Japan) was adopted to perform the real-time quantitative polymerase chain reaction (RT-qPCR).
The expression level of mRNA was quantified using the -ΔΔCt method, and β-actin was taken as the control. Table 1 shows the corresponding primers.

Quantitative real-time polymerase chain reaction (qPCR).
The quantitative analysis of Cullin 5 (Cul5) gene expres-Table II. Primer sequences used for RT-PCR and qPCR analysis. sion was performed using Platinum ® SYBR ® Green qPCR SuperMix-UDG (Invitrogen-Life Technologies) and oligo primers (Invitrogen-Life Technologies) designed for the detection of Cul5 and hypoxanthine-guanine phosphoribosyltransferase (HPRT) housekeeping gene (Table II). The amplification reactions were performed using a DNA Engine Opticon Real-Time Thermal Cycler (Bio-Rad Laboratories). The relative expression of Cul5 mRNA was determined using the ∆∆C T Livak method ( 21) and data were reported as fold increase calculated using the 2 -∆∆CT ± SD equation.