Fig. 2.

Opa1 forms redox sensitive oligomers upon H₂O₂ or ischemia reperfusion in vitro and ex-vivo.

(A) Mouse hearts in a Langendorff system were perfused with oxygenated buffer (Untr), 1 mM H₂O₂ (H₂O₂) or underwent ischemia reperfusion (I/R). Tissues were lysed and equal amounts of protein (20 μg) from cardiac tissues were separated by SDS-PAGE in the indicated reducing or non-reducing conditions. Immunoblotting was performed using the indicated antibody. Red P: membranes were stained using Red Ponceau. Asterisks indicate a ∼180 KDa Opa1 immunoreactive band that disappears in reducing SDS-PAGE; arrowhead a ∼240 KDa Opa1 immunoreactive band that is also sensitive to reduction.

(B) Adult cardiac myocytes were perfused with oxygenated buffer (Untr), subjected to I/R or treated with 1 mM H₂O₂ for 30 min and lysed. Equal amounts of protein (20 μg) were separated by reducing or non-reducing SDS-PAGE and immunoblotted using the indicated antibodies. Asterisks indicate a ∼180 KDa Opa1 immunoreactive band that disappears in reducing SDS-PAGE.

(C) Percentage of oxidized Opa1 calculated by densitometric analysis in non-reducing immunoblots from experiments as in B. N = 5 independent experiments (open dots). Line indicates mean, whiskers SEM. *, p < 0.05 in a one-way ANOVA with Tukey's mean comparison.

(D) MEFs were treated with 1 mM H₂O₂ for the indicated time and lysed. Equal amounts of protein (15 μg) were separated by reducing or non-reducing SDS-PAGE and immunoblotted using the indicated antibodies. Asterisks indicate the ∼180 KDa Opa1 immunoreactive band that disappears in reducing SDS-PAGE; arrowhead the ∼240 KDa Opa1 immunoreactive band also sensitive to reduction.

(E) 2D reducing/non-reducing SDS-PAGE of total lysates (20 μg) of mouse hearts perfused in a Langendorff system with oxygenated buffer (Untr) or 1 mM H₂O₂ (H₂O₂) for 15 min, as indicated. Equal amounts of protein (15 μg) were separated by a 1st dimension reducing or non-reducing SDS-PAGE and the lane was excised and mounted on the top of a 2nd dimension reducing or non-reducing SDS-PAGE. In parallel, an identical 1st dimension lane was transferred onto PVDF membranes and immunoblotted using and anti Opa1 antibody for reference. The top images show this anti Opa1 immunoblot on the 1st dimension reference lane. The bottom images show the corresponding anti Opa1 immunoblot of the 2nd dimension SDS-PAGE ran in the indicated conditions, transferred onto PVDF membranes and immunoblotted using and anti Opa1 antibody. The dotted lines indicate the molecular species immunoreactive for Opa1 in the different 1st and 2nd dimension immunoblots.