Analysis and characterization of PQ modification effect on Spx and PQ modified form of Spx. A. PQ-induced modifications of the CXXC motif of Spx. Strains producing wild-type and CXXC motif mutated Spx-HA fusion proteins were treated with or without 100 μM PQ for 15 min. Crude cell extracts from each strain were analyzed by western blot using an antibody against HA. lane 1–2 (wt): strain BSY2534 producing Spx-HA protein; lane 3–4 (10A): strain BSY2531 producing Spx10A-HA protein; lane 5–6 (13A): strain BSY2533 producing Spx13A-HA protein; lane 7–8 (10A13A): strain BSY2535 producing Spx10A13A-HA protein; lane 9–10: vector control (strain BSY2530 that did not express any HA fusion protein). lane 2, 4, 6, 8, 10: with PQ treatment; lane 1, 3, 5, 7, 9: without PQ treatment. The modified forms of Spx were marked as Spx-M1, Spx-M2 and native forms of Spx were marked as Spx. B. Time course changes of PQ-induced modifications of Spx in vivo. Strain BSY2534 producing Spx-HA was incubated with 100 μM PQ for different time (0–6 hr). 40 μg protein from cell crude extracts obtained at each time point was analyzed by western blot using anti-HA antibody. lane 1: 0 min; lane 2: 15 min; lane 3: 1 hr; lane 4: 2 hr; lane 5:4 hr; lane 6: 6 hr.