BFL-1 is the least studied pro-survival BCL-2 family member, partly due to the lack of high-quality tools, such as antibodies that can detect this protein at endogenous levels. BFL-1 mRNA expression may not mirror BFL-1 levels due to post-translational regulation of this protein [1, 2]. Hence, there is a requirement for a reliable antibody to detect endogenous BFL-1 in cells.

To generate BFL-1-specific monoclonal antibodies, we utilized mice lacking A1 (the mouse homologue of BFL-1) [3, 4]. We hypothesized that A1−/− mice would generate a better immune response against BFL-1 as their immune cells have not been tolerized to the highly homologous mouse A1 protein. Hence, we immunised the mice with a truncated recombinant BFL-1 protein (aa1-151, lacking the C terminal region) and two KLH-conjugated peptides corresponding to the BFL-1 protein regions aa71–84 and aa129–154. One BFL-1 reactive monoclonal antibody identified in an ELISA screen (data not shown) was validated by Western blot analysis of lysates from HEK293T cells overexpressing FLAG-tagged BFL-1 protein [5] (Supplementary Fig. 1). To verify the specificity of the BFL-1 antibody, we examined BFL-1 protein stability in M14 (previously shown to express BFL-1 protein [6]) and SK-MEL30 melanoma cells. As expected, the protein synthesis inhibitor cyclohexamide (CHX) decreased and the proteasome inhibitor (MG132) increased the intensity of the BFL-1 protein band (Supplementary Fig. 2). To test the cross-reactivity of the BFL-1-specific antibody for its murine counterpart A1, lysates from LPS (known to upregulate A1 mRNA [7]) stimulated lung tissues were analysed by Western blotting. Lysates from BFL-1 CRISPR deleted [8] and parental M14 cells served as controls. This demonstrated the reactivity of the monoclonal antibody to human BFL-1 but not mouse A1 (Supplementary Fig. 3).

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