ON-TARGETplus Human EGR1 (1958) siRNA - Set of 4

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 2 × 10^6 cells	siRNA concentration-50–75 nm

Upstream tips
Seed 2 × 10^6 cells
Protocol tips
siRNA concentration-50–75 nm

Publication protocol

Dominant-negative Egr-1 construct (EBGN-EGR-1) expresses a truncated version of murine Egr-1 lacking the transactivational domain and containing only the zinc-finger DNA-binding site (amino acids 322–533) fused to GST. The empty vector, EBGN, contains a nuclear-expressed GST (Al-Sarraj et al, 2005); both these vectors are a kind gift from Professor G Thiel (University of Saarland Medical Center, Homburg, Germany). pEBS14luc, an Egr-1 reporter construct, contains four copies of Egr-1 response element of the Egr-1 gene promoter in the pGL3-promoter vector (also a gift from Professor G Thiel, University of Saarland Medical Center) (Al-Sarraj et al, 2005). To normalise for transfection efficiency, a constitutive Renilla luciferase expressing plasmid was used (pRL-CMV, Promega Corporation, Madison, WI, USA). For transfection, HCT15 cells (2 × 106) were pelleted and resuspended in transfection solution V (Lonza Group Ltd., Basel, Switzerland) containing 2.5 μg of plasmid unless otherwise stated. Transfection was performed by nucleofection using program T13 according to the manufacturer's protocol (Amaxa). GFP plasmid (2.5 μg) was used to determine transfection efficiency, which was 48±7%. Control cells were subjected to the same transfection condition without any plasmids. At 24 h after transfection, cells were resuspended in media and seeded for Annexin V and protein assays. Similarly, stable transfection of Bcl-2 or empty vector (Neo) was carried out in HCT15 cells using the same transfection protocol (a kind gift from Dr Peter Daniel, University of Berlin, Berlin, Germany). Pools of stable clones were selected with 1 μm of G418. siRNA transfection was carried out by the same nucleofection protocol as for plasmids using 50–75 nm siRNA. The following c-FLIP sequences were targeted: c-FLIPS/L1: 5′-GGAGCAGGGACAAGTTACA-3′, c-FLIPS/L2: 5′-GCAAGGAGAAGAGTTTCTT-3′, c-FLIPS/L3: 5′-GAGGTAAGCTGTCTGTCGG-3′ (Nakajima et al, 2008), c-FLIPS1: 5′-CACCCTATGCCCATTGTCC-3′, cFLIPS2: 5′-CATGGAACTGCCTCTACTT-3′ (Zhang et al, 2004; Longley et al, 2006). The GFP target sequence was: 5′-GGCUACGUCCAGGAGCGCACC-3′. To knock down Egr-1, an siRNA Smartpool containing a mixture of four Egr-1-specific siRNAs was used (Dharmacon, Thermo Fisher Scientific, Rockford, IL, USA). Transfection was carried out as for c-FLIP siRNAs.

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