Specificity of the Bak Antibodies Used in This Study. (A) Autoradiography of N-Bak And

Figure S1

Specificity of the Bak antibodies used in this study. (a) Autoradiography of N-Bak and Bak translated in vitro in the presence of 35S-methionine in the rabbit reticulocyte lysate (left filter) and Western blot from the parallel samples translated without 35S-methionine, probed with anti-Bak antibodies (Sigma B5897, middle filter) and re-probed (Upstate 06-536, right filter). Bak (about 25 kD) and N-Bak (about 20 kD) proteins are recognized by both antibodies. The additional bands on the autoradiography (about 15 kD in the N-Bak lanes and about 20 kD in the Bak lane) result from aberrant usage of downstream translation initiation site (methionine 58) that occurs in the reticulocyte lysate.1 These bands lack the N-terminal epitope for the Bak antibodies. However, they are not observed in the HeLa cells transfected with N-Bak or Bak engineered to contain internal hemagglutinin epitope (not shown) and are thus reticulocyte lysate-specific artefacts. N-Bak-L76E is apoptotically inactive mutant of N-Bak.2, 3 (b) Western blot of HeLa cells transiently transfected with N-Bak expression plasmids and probed with anti-Bak antibodies (Sigma B5897). To avoid cell death caused by overexpressed N-Bak, the apoptotically inactive mutant (N-Bak-L76E) was used or the anti-apoptotic Bcl-xL was co-transfected. The antibody recognized transfected N-Bak (about 20 kD) and endogeneous Bak (about 25 kD). Similar results were obtained with antibodies from Upstate Biotechnology (06-536) (not shown).

References

1.  Svitkin YV, Ovchinnikov LP, Dreyfuss G, Sonenberg N. General RNA binding proteins render translation cap dependent. EMBO J 1996; 15: 7147-7155.

2.  Sun YF, Yu LY, Saarma M, Timmusk T, Arumäe U. Neuron-specific Bcl-2 homology 3 domain-only splice variant of Bak is anti-apoptotic in neurons, but pro-apoptotic in non-neuronal cells. J Biol Chem 2001; 276: 16240-16247.

3.  Sun YF, Yu LY, Saarma M, Arumäe U. Mutational analysis of N-Bak reveals different structural requirements for antiapoptotic activity in neurons and proapoptotic activity in nonneuronal cells. Mol Cell Neurosci 2003; 23: 134-143.