Figure 3.

At-TSPO Binds to Heme in Vitro: Evidence from the Pull-Down Assay.

Purified AtTSPO-6His from yeast microsomes was dialyzed to remove the imidazole used in the Ni-NTA purification and then mixed with hemin (A) or PPIX (B) and the sample repurified on the Ni-NTA column. The affinity chromatography eluates of solubilized proteins from EV-transformed yeast or hemin or PPIX alone were used as controls. The absorbance at 405 nm of the eluted Ni-NTA fraction was recorded and normalized to the values for hemin or PPIX alone passed though the Ni-NTA column. Immunoblot analyses (bottom panel in [A] and [B]) were used to assess the presence of At-TSPO in the various fractions. The experiments were repeated at least three times, each in duplicate, and the error bars represent se.