ON-TARGETplus Human PCSK6 (5046) siRNA - Individual

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 2 days at 37°C.

Protocol tips
Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 2 days at 37°C.

Publication protocol

A253 and Detroit 562 (D562) are human head and neck squamous cell carcinoma cell lines, cultured in S-MEM with 10% FBS and 2 mM L-glutamine [20]. For all SiRNA experiments, cells were transfected by Lipofectamine® RNAiMAX Reagent (Invitrogen, Carlsbad, CA) using the manufacturer’s protocol. The following SiRNAs were purchased from Thermo Scientific (Lafayette, CO): ON-TARGETplus Non-targeting siRNA #1 (#D-001810-01-0005) for control siRNA, ON-TARGETplus PCSK7 siRNA (#J-005988-07-0005) for PC7, ON-TARGETplus FURIN siRNA-SMARTpool (#L-005882-00-0005) for Furin, and ON-TARGETplus PCSK6 siRNA (#J-005983-05-0002) for PACE4.

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.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing siRNA / miRNA gene silencing Human - Detroit 562 / D562 PACE4 using ON-TARGETplus Human PCSK6 (5046) siRNA - Individual from Dharmacon.

Manufacturer protocol

Download the product protocol from Dharmacon for ON-TARGETplus Human PCSK6 (5046) siRNA - Individual below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing siRNA / miRNA gene silencing Human - Detroit 562 / D562 PACE4 using ON-TARGETplus Human PCSK6 (5046) siRNA - Individual from Dharmacon. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.