| Cell Line Species/Tissue: |
Rat
/
Vascular Smooth Muscle Cells |
| Transfection Reagent: |
HiPerFect |
Nucleic Acid:
|
siRNA (dsRNA) |
Growth Medium:
|
DMEM/F-12 (no antibiotics) |
Percent Serum (%):
|
10% FCS |
Reporter System:
|
|
Plasmid Purification Method:
|
|
Plate Format:
|
6-well plates |
Number of Cells:
|
|
Percent Confluence(%):
|
70% |
Amount of Nucleic Acid (µg):
|
240 pmol (optimized) |
Amount of Enhancer (µl):
|
|
Amount of Reagent (µl):
|
12µl |
Complex Incubation on Cells (hrs):
|
72h |
Analysis Performed Post-Transfection (hrs):
|
72h |
Transfection Efficiency (%):
|
|
| Knockdown Efficiency (%): |
Protein level 31+/- 10% |
| |
| Any modifications to the protocol?:
|
| |
| Notes: |
| VSMCs were used between passages 5 and 9. The cells at confluence were washed with serum-free medium and then maintained in DMEM/F-12 with 0.1% FCS for 24–48 h. The medium was refreshed before treatment. Small interference RNA (siRNA)-Rsk1 (Rn_Rps6ka1_2_HP siRNA, targeting rat Rps6ka1, gene accession no. NM_031107; target sequence AAG GCC TTT
CTA ATA AAC CTA) and nonsilencing control siRNA (siRNA-Ctl) were obtained from Qiagen. Rat VSMCs were cultured in DMEM/
F-12 with 10% FCS (no antibiotics) in six-well plates for 1 day (reaching ~70% confluence) and then transfected with 240 pmol of
siRNA (an optimized amount) mixed with 12 µl of HiPerFect in 0.6 ml of serum-free DMEM/F-12 per well. After 3 h of transfection, 2.4 ml of DMEM/F-12 with 0.1% FCS was added. The medium was changed next day with 3 ml of DMEM/F-12 with 0.1% FCS and was further cultured for 48 h before IL-1 treatment. The transfection under these conditions did not cause observable changes in cell shape and adherence. |
| References: |
| Ribosomal S6 kinase-1 modulates interleukin-1ß-induced persistent activation of NF-kappaB through phosphorylation of IkappaBß
Shanqin Xu, Hossein Bayat, Xiuyun Hou, and Bingbing Jiang
Vascular Biology Unit, Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts
Am J Physiol Cell Physiol 291: C1336–C1345, 2006. |