Figure 2
From: Bak apoptotic function is not directly regulated by phosphorylation
Phosphotagged Bak variants confirm that IEF conditions can detect phosphorylated Bak. (a) Three tags containing the PKA phosphorylation motif (R/KR/KXpS/pT, underlined) were added to the Bak N-terminus to generate positive controls of Bak phosphorylation. phospho1S→ABak, which lacks serine in the tag, was generated as a negative control. Phosphorylatable serine residues are indicated (bold). pI and MW were calculated at http://web.expasy.org/protparam/, with a serine to glutamate substitution representing phosphorylation. (b) IEF detects phosphorylated Bak variants. Membrane fractions from Bak−/−Bax−/− MEFs expressing the indicated phosphotagged Bak variants were prepared in the presence of sodium orthovanadate, then resuspended in wash buffer, and incubated with or without λ-phosphatase before running on IEF and immunoblotting for Bak. *Phosphorylated species. Note that a lower band in the phospho1S→ABak samples was not evident in other experiments. (c) SDS-PAGE detects phosphorylation of phospho3Bak. Samples prepared for IEF in (b) were supplemented with SDS sample buffer and run on reducing SDS-PAGE before immunoblotting for Bak. Data are representative of three independent experiments
