Rat Anti-BAX Recombinant Antibody (clone 3C10) (CAT#: PABC-255)

Figure 1 Immunoprecipitation

3C10 binds non-activated BaxS184L. Membrane fractions from Bak^−/−Bax^−/− MEFs expressing BaxS184L were immunoprecipitated with the 3C10 or anti-FLAG (negative control) antibodies and immunoblotted for Bax.

Iyer, S., Anwari, K., Alsop, A. E., Yuen, W. S., Huang, D. C., Carroll, J., ... & Kluck, R. M. (2016). Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies. Nature communications, 7, 11734.

Figure 2 Epitope mapping

The 3C10 epitope maps to the Bax α1–α2 loop, close to α1. Immunoreactivity of the rat monoclonal antibody, clone 3C10, towards biotinylated human Bax peptides as determined by enzyme-linked immunosorbent assay. X axis indicates Bax residue number or control reactions. Mean and s.d. of three independent experiments.

Iyer, S., Anwari, K., Alsop, A. E., Yuen, W. S., Huang, D. C., Carroll, J., ... & Kluck, R. M. (2016). Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies. Nature communications, 7, 11734.

Figure 3 3C10 binds WT Bax.

Lysates of Bak^−/−Bax^−/− MEFs expressing Bax (Exp.) or of Bak^−/−Bax^−/− membranes reconstituted with recombinant Bax (100 nM, Rec.) were immunoprecipitated with 3C10 and immunoblotted for Bax.

Iyer, S., Anwari, K., Alsop, A. E., Yuen, W. S., Huang, D. C., Carroll, J., ... & Kluck, R. M. (2016). Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies. Nature communications, 7, 11734.

Figure 4 Antibodies to the BAX a1-a2 Loop can Activate Mitochondrial BAX

Antibodies activate only the mitochondrial S184L form of BAX. Antibodies were incubated with either mouse liver mitochondria supplemented with recombinant BAX or membrane fractions from mouse embryonic fibroblasts expressing BAX S184L. The S/N and mitochondrial (Mito) fractions were examined for cytochrome c. Note that antibodies to the N terminus of the BAX α1-α2 loop are most efficient at activating mitochondrial BAX, while no antibody activated WT BAX. Representative of at least four independent experiments.

Robin, A. Y., Iyer, S., Birkinshaw, R. W., Sandow, J., Wardak, A., Luo, C. S., ... & Colman, P. M. (2018). Ensemble properties of Bax determine its function. Structure, 26(10), 1346-1359.

Figure 5 Antibodies to the BAX α1-α2 Loop can Inhibit Cytosolic BAX

All antibodies either block or retard BAX translocation and cytochrome c release triggered by tBID. Mouse liver mitochondria were incubated with BAX and antibody for 30 min at 37°C, and then with tBID for another 10 or 30 min. The supernatant (S/N) and mitochondrial (Mito) fractions were then examined for cytochrome c and BAX. Representative of three independent experiments.

Robin, A. Y., Iyer, S., Birkinshaw, R. W., Sandow, J., Wardak, A., Luo, C. S., ... & Colman, P. M. (2018). Ensemble properties of Bax determine its function. Structure, 26(10), 1346-1359.