Figure 3. MBP fibers are viable and retain myeloid cell-binding specificity.

(A) Diagram comparing wild type Ad5 and the MBP fibers. The MBP fiber is derived from the 566FF platform that our group has previously developed [20]. Briefly, the knob domain is removed and replaced with the trimerization region from the T4 phage fibritin protein fused to the MBP targeting ligand via a flexible linker ([GGGS]₄). (B) Assessment of MBP fiber viability by Western blot. 293T cells were harvested 48 h after transfection with expression plasmids for wild type Ad5, MBP, or inverted MBP (iMBP) fibers. Protein supernatants from 293T cells were incubated at either 95°C (boiled, B) or room temperature (unboiled, U) prior to SDS-PAGE separation. Unboiled samples demonstrate that the majority of fibers are trimerized. (C) Evaluation of fiber binding to CD11b⁺ (myeloid) BMCs from wild type (C57BL/6) and transgenic mice that express hCAR (hCAR Tg). 293T protein supernatants from B were added to BMCs and fiber-bound CD11b⁺ cells were detected by flow cytometry. A representative experiment is shown for C.