Characterisation of a novel A1-specific monoclonal antibody

Abstract

Dear Editor, A1/BFL-1 is the least studied pro-survival BCL-2 family member. This can be largely attributed to the lack of proper tools to study A1/BFL-1 function. Owing to the genomic organisation of the A1 locus in mice (three expressed A1 genes and one pseudo-gene, interspersed by unrelated genes) 1 a knockout is challenging. We generated shRNA transgenic mice in which all functional A1 isoforms were knocked down. In accordance with A1 mRNA expression studies, we found that A1 is critical for the development and survival of lymphocytes and granulocytes. 2 As the A1/ BFL-1 protein is regulated by ubiquitin-dependent proteasomal degradation, the A1 mRNA expression data may not truly reflect the A1/BFL-1 protein levels. Previous attempts to generate A1-specific antibodies have failed and commercially available antibodies do not reliably detect the endogenous protein.To generate A1-specific monoclonal antibodies, we immunised rats with a truncated/mutated A1 protein (delta-C20, P104K) 3 together with two KLH-conjugated peptides corresponding to central and C-terminal residues of the A1 protein (aa71-84; aa129-154). Screening by ELISA and western blotting identified one monoclonal antibody that detected overexpressed A1-a, A1-b and A1-d, and to a lesser extent overexpressed human homologue BFL-1 (data not shown and Figure 1a). To test whether this antibody could reliably detect endogenous A1, we used the mouse WEHI-231 B lymphoma cells, known to express high levels of this protein. 4 Western blotting revealed a single band of the molecular weight expected for A1 in untreated WEHI-231 cells (Figure 1b, first lane). Overexpressed A1 protein is highly unstable due to ubiquitin-dependent proteasomal degradation. 5 To further verify the specificity of the A1 antibody, we tested the impact of protein synthesis inhibition or proteasome inhibition on the protein detected in WEHI-231 cells. As expected, the protein synthesis inhibitor cyclohexamide (CHX) decreased the intensity of the protein band, whereas the proteasome inhibitor (MG132) increased it substantially (Figure 1b). Furthermore, we were able to show that this antibody can be used to immunoprecipitate endogenous A1 protein from lysates of WEHI-231 cells (Figure 1c). Next we examined whether this antibody could also detect endogenous A1 in primary mouse cells. In accordance with previous reports on A1 mRNA expression, 1 we could reliably detect A1 protein in haematopoietic tissues, such as the lymph nodes and spleen but not in the heart, kidney, liver or lungs (Figure 1d). Immunohistochemical staining using this antibody showed strong A1 protein staining within cell foci in the germinal centres of lymph nodes of non-immunised mice (Figure 1e). No staining with this antibody against A1 was observed in nonhaematopoietic tissues, such as the pancreas or the heart (data not shown). To further validate the specificity of this A1 antibody in primary cells, mouse spleen cells were treated with crosslinking IgM antibodies, a stimulus known to upregulate A1 mRNA levels in...

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

“…34 In wild-type cells, A1 was markedly upregulated by 4 h of stimulation, whereas no protein was detected in A1-deficient cells. This analysis showed no detectable compensatory upregulation of the other pro-survival BCL-2 family proteins, BCL-2, BCL-XL and MCL-1, in A1-deficient cells (Figure 1b).…”

Differential effects of Vav‐promoter‐driven overexpression of BCLX and BFL1 on lymphocyte survival and B cell lymphomagenesis

“…Twenty to thirty micrograms of total protein was loaded on 12% Bis‐Tris acrylamide gels and blotted on Amersham™ Hybond™—ECL nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). The following antibodies were used for protein detection: rabbit anti‐BFL1 (kindly provided by Jannie Borst 50), rat anti‐mouse A1 (WEHI, 6D6, 2 μg·mL −1 ) 51, rabbit anti‐BIM/BOD (Enzo, Farmingdale, NY, USA, polyclonal, ADI‐AAP‐330‐E, 0.2 μg·mL −1 ), rabbit anti‐MCL1 (polyclonal, Rockland, Pottstown, PA, USA, Cat# 600‐401‐394, 2.2 μg·mL −1 ), rabbit anti‐BCLX (54H6, Cell Signaling, Danvers, MA, USA, Cat# CS2764, 1 : 1000), mouse anti‐BCL2 (7/Bcl‐2, BD Biosciences, 0.5 μg·mL −1 ), mouse anti‐HA (HA.11, Covance, Princeton, NJ, USA, 1 : 1000), rabbit anti‐MYC (Y69, Abcam, Cambridge, UK, ab32072), rabbit anti‐VAV1 (Cell Signaling #2502, 1 : 1000), rabbit anti‐gamma‐H2A.X (Ser139, Cell Signaling #2577, 1 : 1000), rabbit anti‐GAPDH (14C10, Cell Signaling #2118, 1 : 5000), rabbit anti‐beta‐Actin (polyclonal, Cell Signaling #4967, 1:1000) and mouse anti‐HSP90 (F‐8, Santa Cruz, Dallas, TX, USA, Cat# sc‐13119, 0.2 μg·mL −1 ). All primary antibodies were diluted in 5% BSA in PBST and blots were incubated overnight at 4 °C.…”

The BCL-2 pro-survival protein A1 is dispensable for T cell homeostasis on viral infection

“…The lack of an obvious phenotype was unexpected, as the levels of A1 mRNA and protein were both rapidly increased in T cells on TCR/CD3 ligation or stimulation with mitogens 17,18,23 A1 induction has been defined as a central step in rewiring the cell survival machinery in T cells during the transition from a resting to an activated state. This is associated with a change from a cytokine receptor (mainly IL-7R) to a TCR-driven survival programme and includes the induction of BCL-XL and A1, accompanied by a downregulation of BCL-2.…”

“…Indeed, it has been shown before that A1 is tightly regulated by ubiquitin-dependent proteasomal degradation. 18,35 Future experiments of A1 protein stability in Tregs will show whether this hypothesis holds true. Interestingly, non-challenged A1 −/− mice displayed a slight reduction in Tregs compared with wild-type mice.…”

“…However, these observations might not be totally surprising, given that A1 protein levels are very low in unstimulated cells. 18 Therefore, we investigated a role for A1 in mice that had been challenged with different viral infections. These studies clearly demonstrate that loss of A1 alone does not impair T cell-driven immune responses.…”