superscript iii first strand complementary dna synthesis kit  (Thermo Fisher)

Journal:

Article Title: Grape seed extract inhibits VEGF expression via reducing HIF-1α protein expression

doi: 10.1093/carcin/bgp009

Figure Lengend Snippet: GSE inhibits HIF-1α expression. (A) GSE inhibits HIF-1α protein expression. U251 cells were incubated under normoxia, 1% oxygen or treated with DFX for 16 h, in the absence or presence of increasing concentrations of GSE (6.5–26 μg/ml). Whole-cell lysates were analyzed by immunoblotting with antibody against HIF-1α. β-Actin level was used as a loading control. (B) Silence of HIF-1α expression resulted in a decrease in VEGF expression. HIF-1α siRNA and control siRNA were transfected into U251 cells expressing VEGF reporter gene. Cells were incubated under normoxia and hypoxia (1% oxygen) for 24 h and then measured for Luc activity. Data were represented as a ratio to control treated with water in each condition, **P < 0.005. (C) GSE has no apparent effect on HIF-1α mRNA expression. U251 cells were incubated with GSE (10 μg/ml) in the presence of DFX for 16 h. Cells were harvested for HIF-1α mRNA expression by RT-PCR. Data were normalized to β-actin and expressed as a ratio to the control treated with water alone. (D) GSE has little effect on HIF-1α degradation. Cells were first incubated in the presence of DFX (250 μM) for 4 h and then treated with CHX (10 μM) in the presence or absence of GSE (26 μg/ml) for indicated times. Cells were harvested and whole-cell lysates were analyzed for HIF-1α protein level by immunoblotting. (E) GSE inhibits HIF-1α protein synthesis. Cells were pretreated with GSE (26 μg/ml) for 1 h prior to the addition of proteasome inhibitor MG-132 (10 μM). Cells were then incubated for various times as indicated. Cells were harvested and whole-cell lysates were analyzed by immunoblotting for the presence of HIF-1α. (F and G) Relative levels of HIF-1α protein in (D) and (E) were determined by measuring the density of the HIF-1α protein band and normalized to that of β-actin (F) or β-tubulin (G). Data were expressed as a ratio to control at time zero (F) and were the mean ± SD of three experiments. *P < 0.05 versus control treated with water.

Article Snippet: Complementary DNA was synthesized from 1.0 μg of total RNA using the Superscript III first-strand complementary DNA synthesis kit (Invitrogen, Carlsbad, CA) in a final volume of 20 μl with 0.25 μg random hexamer and 200 U of Superscript RNase H reverse transcriptase.

Techniques: Expressing, Incubation, Western Blot, Transfection, Activity Assay, Reverse Transcription Polymerase Chain Reaction