ON-TARGETplus Human PCSK7 (9159) siRNA - Individual

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate cells for 1–3 days at 37°C.

Protocol tips
Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate cells for 1–3 days at 37°C.

Publication protocol

Cell lysates were harvested two days after siRNA transfection and subjected to Western blot analysis as described [33]. For CoCl2 and PPP treatments, the transfected or non-transfected cells were incubated with drugs overnight. The treatment time with MG-132 was 30 min. For CHX treatment, the cells were incubated for different times as labeled in the corresponding figures. Regarding VEGF-A and VEGF-C analysis, the cells were first transfected with SiRNA for 2 days and serum-free media were collected after 1-day incubation. The conditioned media were concentrated with Microcon centrifugal filters (#42407) from Millipore (Billerica, MA) and used for Western blot analysis. The following antibodies were used: HIF-1α (#610958) from BD Biosciences (San Jose, CA), VEGF-A (#sc-152), VEGF-C (#sc-1881) and IGF-1Rβ (#sc-713) from Santa Cruz Biotechnology (Santa Cruz, CA), and GAPDH (#ab8245) from Abcam (Cambridge, MA). The quantification of Western blot results was performed by using ImageJ developed by the National Institutes of Health.

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Manufacturer protocol

Download the product protocol from Dharmacon for ON-TARGETplus Human PCSK7 (9159) siRNA - Individual below.

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