CYB5D2 Requires Heme-Binding to Regulate HeLa Cell Growth and Confer Survival from Chemotherapeutic Agents
Figure 3
CYB5D2 binds to type b heme, and exists in both oxidized and reduced forms.
A) Purified recombinant GST-CYB5D2 and GST-CYB5D2(D86G) were thrombin-cleaved, and the uncleaved and cleaved (CYB5D2 and CYB5D2(D86G), respectively) proteins were separated by SDS-PAGE followed by Western blot analysis for CYB5D2 using anti-CYB5D2 antibody. B) Recombinant GST-free CYB5D2 and CYB5D2(D86G) proteins were oxidised by K3Fe(CN)6, followed by scanning at the indicated wavelength range using a spectrophotometer. The peak absorbance for CYB5D2 was observed at approximately 559 nm. C) Recombinant GST-free CYB5D2 and CYB5D2(D86G) proteins were reduced by sodium dithionite treatment and scanned for their absorbance peaks within the indicated wavelength range. The maximum absorbance peaks for the oxidized and reduced forms of human CYB5D2 were 402 nm and 420 nm, respectively.