control shrna plasmid containing a scrambled shrna sequence  (Millipore)

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: RNAi mediated down-regulation of IRAK1 expression. HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence or an IRAK1 specific shRNA. Geneticin selected cells were treated with medium only (Med) or 10 ng/ml IL-1β. After 24 hours cells were lysed in PBS with 2 M urea and 0.85% SDS and proteins examined by Western blotting. IRAK1 and GAPDH were detected using polyclonal anti-IRAK1 and anti-GAPDH rabbit antibodies, respectively.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Expressing, Transfection, Construct, Negative Control, shRNA, Sequencing, Western Blot

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: IRAK1 is required for CC chemokine secretion. (A and B), HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence (open bars) or IRAK1 specific shRNA (filled bars). (C and D), cells were transfected with either an empty (open bars) or IRAK1(1-217) (filled bars) expression construct. Geneticin selected cells were treated with medium only, 10 ng/ml IL-1β or 50 ng/ml TNF-α for 24 hours. Secretion of CCL5 (A and C) and CCL20 (B and D) was determined by ELISA. Expression of CCL5 and CCL20 in the control cells (open bars, scrambled shRNA (A and B) or empty vector (C and D)) was defined as 100 pct. Expression of CCL5 and CCL20 in the IRAK1 targeted cells (filled bars, IRAK1 specific shRNA (A and B) or IRAK1(1-217) expression vector (C and D)) is graphically represented relative to the control cells. * p < 0.05 compared to control cells transfected with scrambled shRNA plasmid (A and B) or empty vector (C and D) and receiving the same treatment.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Transfection, Expressing, Construct, Negative Control, shRNA, Sequencing, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: IL-1β enhancement of IFN-γ induced CXC chemokine expression is IRAK1 independent. A, HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence (open bars) or IRAK1 specific shRNA (filled bars). B, cells were transfected with either an empty (open bars) or IRAK1(1-217) (filled bars) expression construct. Geneticin selected cells were treated with 20 ng/ml IFN-γ or 20 ng/ml IFN-γ plus 10 ng/ml IL-1β for 2 hours. Expression of CXC chemokine mRNAs was examined using real-time RT-PCR. Expression of the individual CXC chemokine mRNA in response to co-treatment with IFN-β+ IL-1β is graphically represented relative to expression induced by IFN-γ alone. * p < 0.05 compared to cells transfected with the same vector and treated with IFN-γ only. ** p < 0.01 compared to cells transfected with the same vector and treated with IFN-γ only. # p < 0.05 compared to control cells transfected with empty vector and receiving the same treatment.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Expressing, Transfection, Construct, Negative Control, shRNA, Sequencing, Quantitative RT-PCR, Plasmid Preparation, Control