FIG. 3.

Recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5α proteins exhibit similar cross-linking patterns. (A) Western blot (anti-FLAG) showing purified monomeric (M) and dimeric (D) proteins cross-linked with increasing concentrations of EGS (lanes 3 to 8) or controls without EGS (lanes 1 and 2). (B) Western blots (anti-FLAG) showing purified recombinant TRIM5-21R dimer added to 293T cell lysate and cross-linked in the presence of increasing concentrations of EGS (first panel); TRIM5-21R expressed in 293T cells, cross-linked directly in the lysate, and concentrated by StrepTactin affinity purification (second panel); and TRIM5α expressed in 293T cells, cross-linked directly in the lysate, and concentrated by α-HA affinity purification (third panel). Lanes 1, 5, and 9 show controls in the absence of EGS, and the asterisk denotes contaminating immunoglobulin G heavy chain eluted from the HA-Sepharose beads. Note that all of the samples were run on the same gel, and migration positions can therefore be compared directly.