Fig. 6. Mammalian PKC-, -, - or - co-expression in S. cerevisiae differentially interferes with acetic-acid-induced Bcl-xL phosphorylation. (A) Western blot analysis reveals two different Bcl-xL migrating bands in protein extracts obtained from yeast expressing Bcl-xL and treated with 300 mM acetic acid for 1 hour. The intensity of these two Bcl-xL bands is dependent on the PKC co-expressed isoform. Without acetic acid, only a single band of Bcl-xL at 9 kDa can be observed (see Fig. 8B); the lane corresponding to protein extracts obtained from yeast co-expressing PKC- and Bcl-xL was located in a different part of the same gel. (B) In vitro phosphatase treatment of protein extracts obtained from yeast expressing Bcl-xL and treated with 300 mM acetic acid for 1 hour. Yeast extract with 25 µg protein was incubated with 400 U -protein phosphatase (+-PPase), at 30°C for 30 minutes. Yeast extract without -PPase was used as a control. Western blot analysis showed that the slow-migrating Bcl-xL band (32 kDa) was completely abolished by -PPase, indicating that this band corresponds to a phosphorylated form of Bcl-xL (P-Bcl-xL). For western blot analysis, protein extracts were separated by 15% SDS-PAGE. For Bcl-xL detection an anti-Bcl-xL goat polyclonal antibody, followed by horseradish-peroxidase-conjugated rabbit anti-goat IgG, were used. For ß-actin detection, used as a loading control, membranes were stripped and then reprobed with an anti-actin rabbit antibody. Immunoblots were developed by enhanced chemiluminescence.