Fig. 5.

Determination of antibody specificity to the proteins expressed from cDNA. A: HEK293T cells were transfected with a pMIG vector (V) or the vector containing a hRSK4 cDNA (K4) starting from ATGβ. Western blot of the cell lysates using the indicated antibody detects multiple proteins at 90- (arrowhead), 72- and 55-kD that are much more abundant in the cDNA transfectants than in the vector counterpart, although the blot for β-actin shows that a less amount of protein from the cDNA transfectants was loaded. B: Transfection of Hela cells with the same hRSK4 cDNA in a pcDNA3.1 vector also results in multiple proteins detected by several antibodies, compared with non-transfected or vector-transfected Hela cells. Cells transfected with the intact mRSK4 that starts from ATGγ or with its Δ15ntΔE24 variant expressed the 90-kD (arrowhead) and a 37-kD proteins that were detected by ab57231 and JS-31 antibodies. C: MCF15 cells were transfected with the intact hRSK4 and the intact mRSK4 cDNA, with the empty pcDNA3.1 vector as control. Cell lysates were immunoprecipitated with one antibody as indicated (1st and 2nd panels) or with a cocktail of N20, C20, ab57231, ab42100 and JS-30 antibodies (3rd panel), followed by immunoblotting with the same or different antibody as indicated. Note that the 90-kD protein was precipitated in all three blots while multiple proteins were precipitated in the first two blots. Nonspecific immunoglobin and protein-A/G agarose beads, included as negative control, also pull down some 55-kD proteins (arrow).