Figure 7. Effect of Rottlerin on cytosolic and plasma membrane bound PKC isoenzymes.

Isolated cytosol and plasma membranes from untreated (−) or PMA and Rottlerin treated (+) THP-1 cells were subjected to Western blotting using PKC specific antibodies. The amount of total protein loaded (PL) to each well is shown. As an additional loading control, ERK2 was used. The diagram under each blot shows the relative amounts (mean ±s.d.) of the PKC isoenzymes in cytosol and membranes, where the results were normalised against the untreated cytosol and membrane controls. All results are based on equal protein loading and in all cases n = 2 except for PKC δ and υ (PKD3) where n = 3. The arrowhead shows the active PKD3 at 100 kDa, while the two bands with reduced molecular size may be truncated variants of PKD3. The position of the molecular mass markers at 100 and 80 kDa are shown at the left. In order to show the increased level of PKC ε in the cytosol from PMA treated cells (353 µg/well) in the presence of Rottlerin compared to the absence of this compound, a largely increased developmental exposure time of the membrane in the presence of the Luminol substrate was needed.