Figure 3: Detection of oxidatively modified proteins following plasma exposure.
(A) HaCaT cells were exposed to plasma treatment (He, He-O2 and He-N2) for 5 min with 1 hr post-treatment storage and analyzed at different times after plasma treatment. To detect modified protein cells oxidatively, extracts were treated with 2, 4-dinitorphenylhydrazine to derivatize protein carbonyls and then evaluated by Western-blot analysis using 2, 4-dinitrophenyl antibodies and the blot were quantified using Image J. (B) Fluorescent detection of oxidized proteins. HaCaT cells were exposed to plasma treatment (He, He-O2 and He-N2) for 5 min with 1 hr post-treatment storage and analyzed at 24 hr after plasma treatment. Cells were lyzed and evaluated by SDS-PAGE (4–20%) pattern of carbonylated proteins pre-labeled with C5Hz. As a positive control, HaCaT cells were treated with 500 μM H2O2 for 1 hr and analyzed 24 hr later. (C) Total proteins post-stained with ProteinGOLDTM. (D) Semi-quantification of carbonylated proteins were performed by densitometric analysis, expressed as relative values (normalization to total protein) and shown as mean ± S.D (n = 3) and analyzed using Student’s t-test; *P < 0.05.
