Aip1 stimulates the production of low molecular mass actin species. (A) Intermediates from in vitro F-actin disassembly reactions were captured by specific cross-linking between actin subunits along the protofilament axis. Low molecular mass intermediates are marked as monomer, dimer, and trimer. The amount of each species was quantified relative to the reaction with no cofilin or Aip1, and the results are presented in the graph. Quantification is the mean of three independent in vitro reactions run on 4–12% gradient gels stained with Sypro ruby. (B) Equal amounts of cross-linked cell lysates from act1-Q41C and aip1Δ act1-Q41C cells were loaded in duplicate on nonreducing and reducing SDS-PAGE gels, and actin levels were assessed using antiyeast actin antibodies to show monomer and total actin levels, respectively. Blots were probed for Pgk1p as loading controls. Immunoblot signals are from IRDye-labeled secondary antibodies acquired with an infrared imaging system (Odyssey). Actin monomer concentration was quantified after normalization to the Pgk1 signal. Actin levels are normalized to actin monomer levels in extracts from act1-Q41C cells, and the results of four independent cross-linking reactions are presented in the graph. Error bars represent SD.