Figure 2

In vitro direct interaction of Nrf1 with MCRS2. A, binding of MCRS2 by various GST-Nrf1 deletion mutants is shown. The GST protein bead used is indicated above for each track of the autoradiogram. Top panel, in vitro translated MCRS2 was incubated with GST (lane 3) or GST-Nrf1 derivatives (lanes 4–7) beads at 4°C for 2 hours and then was eluted with 0.1 M reduced glutathione. The autoradiography from the eluted supernatants was separated on a 10% SDS-PAGE gel. Lane 1 indicates 1/20 labelled MCRS2 (S³⁵-MCRS2). Middle panel, the same amounts of purified GST (lane 3) and GST-Nrf1 derivatives (lanes 4–7) were used in a GST pull-down assay, separated on a 10% SDS-PAGE gel, and then stained. Bottom panel, diagram of the purified GST and GST-Nrf1 derivatives. B, binding of Nrf1 by various GST- MCRS2 deletion mutants is shown. The GST protein bead used is indicated above each track of the autoradiogram. Top panel, in vitro translated Nrf1 was incubated with GST (lane 3) or GST-MCRS2 derivatives (lanes 4–7) beads at 4°C for 2 hours and was then eluted with 0.1 M reduced glutathione. The autoradiography from the eluted supernatants was separated on a 10% SDS-PAGE gel. Lane 1 indicated 1/60 labelled Nrf1 (S³⁵-Nrf1). Middle panel, the same amounts of purified GST (lane 3) and GST-MCRS2 derivatives (lanes 4–7) were used in GST pull-down assay, separated on a 10% SDS-PAGE gel, and then stained. Bottom panel, diagram of the purified GST and GST-MCRS2 derivatives.