Fig 2: Expression levels of MK2, MK3, and MK5 in HEK293 cells. (A) Total RNA was reverse transcribed and MK2, MK3, MK5, and rRNA transcripts were amplified by PCR with specific primers. PCR products were visualized by electrophoresis on an agarose gel in the presence of ethidium bromide. A size marker (in base-pairs; bp) was run on the gel to confirm the correct size of the PCR products. rRNA was used as an internal control [33]. (B) The relative amounts of MK2, MK3, and MK5 in HEK293 cells was determined by western blotting. The band corresponding to the molecular mass of MK2, MK3, and MK5 is indicated by an arrow. To assure equal sample loading, the blot was stripped and re-probed with antibodies against actin. The molecular mass (in kD) of a protein marker is shown.

Fig 3: MK5 is implicated in PKA-induced HSP27 phosphorylation, while MK2 mediated p38MAPK-triggered HSP27 phosphorylation. (A) Top panel: cells were transfected with scrambled siRNA (sc siRNA; lanes 2-4) or siRNA directed against MK5 (MK5 siRNA, lanes 4-6) or MK2 (lanes 7-9). After transfection, cells were left untreated (-, lanes 1, 4 and 7) or treated for 30 min with 250 µM sodium arsenite (SA, lanes 2, 5 and 8) or 10 µM forskolin (FSK, lanes 3, 6 and 9) and the cells were harvest. Protein levels were examined by western blot. Equal loading was verified by examining the expression levels of actin. M = protein molecular mass marker (in kD). The intensity of the bands was determined by densitometry and the value obtained for phosphorylated Flag-tagged-HSP27 (respectively phosphorylated endogenous HSP27) was arbitrary set as 1.0 and the other values were related to this. A representative experiment is shown and similar results were obtained in two independent experiments. Bottom panel: relative fold induction of HSP27 phosphorylation at Ser-78 in untreated and treated cells that had been transfected with scrambled (sc) or MK5-targeting siRNA. The phosphoHSP27 levels in untreated, scrambled siRNA transfected cells were arbitrary set as 1.0. The bars represent the average (+standard deviation) of three independent results. A significant difference (*p < 0.05; student's t-test) was observed for forskolin-induced HSP27 phosphorylation in scrambled siRNA and MK5-targeting siRNA transfected cells. (B) As in (A), but cells were transfected with siRNA against MK3. RDU values were calculated as in (A). (C) Overexpression of active MKK6 plus p38MAPK (lane 3) or exposure of cells to 250 µM sodium arsenite (SA) for 30 min (lane 5) causes phosphorylation of MK2, while the catalytic subunit of PKA (Ca) or forskolin (FSK) failed to provoke HSP27 phosphorylation. Control cells were transfected with the empty expression vector pRcCMV (lane 1). The molecular mass (in kD) of the protein marker is shown (lane M). The densitometry values obtained for the signals of phosphorylated HSP27 or MK5 (respectively MK2 or MK3) were determined and corrected for the values obtained for actin. RDU values in control cells (lanes 1) were arbitrarily set as 1.0 and the other values were related to this.