Streptavidin-biotin–binding assay.

Biotin-conjugated JH4 was produced by chemical synthesis (see the Chemical Synthesis section in the Supplemental materials). To determine the location of JH4-biotin, cells were incubated with biotin or biotin-JH4 for the indicated durations (Figure 3) and fixed with 2% PFA. After permeabilization, blocking with BSA, and prereaction with H₂O₂ to eliminate endogenous HRP activity, cells were incubated with streptavidin-HRP (Sigma-Aldrich) and DAB solution (Vector Laboratories). To investigate the direct binding of JH4 and progerin, cells lysates, precleared by streptavidin-bead, were incubated with biotin-JH4 or biotin and streptavidin-bead, sequentially. The precipitated materials were subjected to SDS-PAGE and Western blot analysis.