Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells
Fig 1
Validation of MT-3 protein interactions by MT-3 mediated pulldown and V5-mediated co-immunoprecipitation.
(A). Zn7-MT-3 cross-linked agarose beads pulldown proteins isolated from cultured human proximal tubule cells (HK-2) include β-actin (a), tropomyosin 3 (b), enolase 1 (c), and aldolase A (d). (B). MT-3 interacts with myosin-9 (a), β-actin* (b) and enolase 1 (c) in V5-tagged MT-3 expressing proximal tubule cells. To verify the binding of MT-3 to myosin-9, β-actin, and enolase 1 in vitro, the V5 antibody was cross-linked to agarose beads through aldehyde linkage and incubated with HK-2 MT-3-V5 cell lysates. Bound proteins were eluted and detected by western blotting. The corresponding protein was not detected in non-transfected control (no V5 tagged MT-3), no antibody control, and no lysate control. *Note: Heat causes Protein A/G to come off the magnetic beads, and the reducing agent dissociates the heavy and light chains of the antibody, resulting in detection via secondary antibody reactivity towards protein A/G.