Validation of UMN-13 as a custom rabbit anti-human APOBEC3A mAb.

(A) Schematic of human A3A, A3B, and A3G indicating the unique N-terminal epitope used here to generate the A3A-specific mAb UMN-13. The schematic also shows the C-terminal epitope used previously to generate the versatile 5210-87-13 mAb that recognizes these three enzymes. (B) Comparative immunoblots of whole cell extracts from 293T cells expressing each of the 7 human A3 family members with C-terminal HA tags. The blot was probed first with our custom rabbit anti-human A3A mAb UMN-13 (left) and, after stripping, a commercial anti-HA mAb as an expression control (right). The positions of the full-length proteins are indicated by red asterisks. (C) Comparative immunoblots of whole cell extracts from the monocytic cell line THP-1 and a clonal derivative lacking A3A-through-A3G (ΔA-G), each treated with DMSO as a control or LPS/IFN-α to induce expression of multiple A3s including A3A and A3G. The UMN-13 mAb blot on the left shows a single band representing full-length A3A (starting at Met1), which is absent in the deletion mutant, and the 5210-87-13 mAb blot on the right shows A3G (strong top band), A3B (weak band just below A3G), and both A3A translation products (strong band for full-length A3A starting at Met1 and a faster-migrating band for the shorter isoform starting at Met13), which are all absent in the deletion mutant. (D) IF microscopy images of 293T cells expressing A3A-mCherry, A3B-mCherry, or A3G-mCherry. Only the A3A construct is detected by the UMN-13 mAb as indicated by green signal in the same cells and cellular compartments as the A3A-mCherry signal.

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