Journal: Journal of Biomedical Science
Article Title: SB203580 increases G-CSF production via a stem-loop destabilizing element in the 3’ untranslated region in macrophages independently of its effect on p38 MAPK activity
Figure Lengend Snippet: SB203580 enhances G-CSF expression in LPS-treated macrophages. RAW264.7 cells ( a and b ) and BMDMs ( c and d ) were incubated with DMSO (vehicle) or 10 μM SB203580 for 30 min, then 100 ng/ml of LPS was added and incubation continued for 0 to 16 h, then G-CSF protein levels in the medium were measured by ELISA ( a and c ) and G-CSF and IL-1β mRNA levels were determined by RT-PCR and analyzed by gel electrophoresis ( b and d ). ( e ) RAW264.7 cells were incubated with DMSO or 1, 5, or 10 μM SB203580 for 30 min, then LPS (100 ng/ml) was added and incubation continued for 6 h, then G-CSF and IL-1β mRNA levels were determined by RT-PCR ( upper panel ). Levels of G-CSF mRNA were determined by RT-qPCR ( lower panel ), normalized to GAPDH mRNA levels, and expressed relative to the value in the cells treated only with LPS, set as 1. ( f ) THP-1 macrophages were pretreated with DMSO or 10 μM SB203580 for 30 min, then LPS or PBS was added and incubation continued for 0 to 6 h. Total RNA was then isolated and levels of G-CSF and GAPDH (internal control) mRNA determined by RT-PCR and analyzed by gel electrophoresis. The results in ( a ) and ( e ) are the mean ± SD for three independent experiments. * p
Article Snippet: Levels of G-CSF and GAPDH mRNA were determined either by semi-quantitative PCR (RT-PCR) or by real-time quantitative PCR (RT-qPCR) on an iCycler instrument (BioRad, Hercules, CA, USA) as indicated.
Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Quantitative RT-PCR, Isolation