All shRNAs plasmids were purified from the
MISSION shRNA Human Library (Sigma) following manufacturer’s instructions. As control the non-targeting control shRNA (shCo)
MISSION pLKO.1 puro (Cat# SHC001) was included.
siRNAs directed against ERK3 were purchased from Qiagen.
siMAPK6#1 (siERK3#1)
FlexiTube siRNA 5 nmol, siRNA Name: Hs_MAPK6_5, Cat# SI00606025
Sense strand: 5’-
AGUUCAAUUUGAAAGGAAATT-3’
siMAPK6#2 (siERK3#2)
FlexiTube siRNA 5 nmol, siRNA Name: Hs_MAPK6_6, Cat# SI00606032 (siRNA used only in
Figure 4—figure supplement 2B)
Negative control siRNA (siCo) Cat# 1027310.
Cells were seeded one day before transfection at an initial density of 2 × 10
5 cells/well in 12-well plates or at 3 × 10
5 cells/well (6-well plates). Cells were transfected using SAINT-sRNA transfection reagent (SR-2003, Synvolux) according to the manufacturer’s instructions. Unless otherwise indicated, medium was exchanged 24 hr post-transfection for medium without FBS and 48 hr post–transfection supernatants were harvested for IL-8 levels measurement by ELISA. In addition, the knockdown was verified by Western blot and/or quantitative real-time PCR.
Bogucka K., Pompaiah M., Marini F., Binder H., Harms G., Kaulich M., Klein M., Michel C., Radsak M.P., Rosigkeit S., Grimminger P., Schild H, & Rajalingam K. (2020). ERK3/MAPK6 controls IL-8 production and chemotaxis. eLife, 9, e52511.
