(A) Shows the RP-HPLC trace of A1A3 cell extract (from 108 cells) and 50 μg of synthetic RC-101.
(B) Western blot of A1A3 HPLC fractions (23–28 min) using rabbit anti-RC-101 antibody. The arrows indicate the multimeric forms of retrocyclin observed in A1A3 fractions.
(C) TZM-bl cells were infected with HIV-1 (p24 = 2 ng/ml) in the presence or absence of pooled A1A3 fractions (final dilution 1:6 in D10) or 2 μg/ml of RC-101 for 24 h. Infection was quantified by luciferase measurement (average RLU of vehicle control with virus = 85,450).
Error bar represents SEM and n = 3–6; *, p < 0.0015; **, p <0.0001. MALDI-TOF MS spectra of Lys-C digested (D) synthetic RC-101_2K, (E) synthetic RC-101, and (F) A1A3 HPLC fraction 26 reveal that A1A3 cells produce RC-101.