Limited sensitivity of commercially available antibodies for endogenous mouse TRAF1 detection in isolated immune
Ibrahim Ridany1, Ali A Abdul-Sater2
1Department of Biology, York University, Toronto, ON, Canada.
Abstract:
TRAF1 is an NF-κB inducible signaling adaptor protein that regulates immune responses. While it limits the NF-κB and MAPK pathways downstream of TLR in monocytes and macrophages, it promotes the survival of T and B lymphocytes downstream of 4-1BB and CD40, respectively. This multifaceted role requires genetic and pharmacological manipulation of TRAF1 expression and its interaction with its signaling partners. Key to such studies is the availability of reliable and specific antibodies that detect TRAF1 protein expression. Here, we systematically evaluate four different commercially available TRAF1 antibody clones (1F3, H3, E12, and 45D3) using wildtype (WT) and Traf1 knockout (KO) cells as definitive controls. We confirm robust induction of Traf1 mRNA in wild-type bone marrow derived macrophages (BMDMs) following stimulation with LPS, Pam3CSK4, or Resiquimod, and T cell activation by CD3 or CD3 + CD28 co-stimulation. Despite this, clones 1F3 and 45D3 produced non-specific signals in negative-control lysates, while clones E12 and H3 detected mouse TRAF1 only when overexpressed in HEK293FT cells and failed to detect endogenous TRAF1 in isolated BMDMs or T cells, even when lysates were intentionally overloaded or enriched by immunoprecipitation. Notably, clone H3 reproducibly detected TRAF1 in lysates of total splenocytes activated with anti-CD3 or anti-CD3 plus LPS, indicating that the assay itself is functional and that detection becomes possible in mixed cell populations where higher-expressing subsets are present. These findings reveal a critical sensitivity gap: currently available commercial antibodies are not adequate for measuring endogenous TRAF1 in isolated monocytes/macrophages or T cells, the very cell types most relevant to inflammation-driven and autoimmune diseases, and underscore the need for new, rigorously validated reagents to support cell-type specific studies of TRAF1 biology in mouse models.
