Fig 1.
The decrease in OD320nm over time in spectrophotometric ATPase activity assays.
(A) Assays of 2 μg of bovine heart mitochondria with and without 62.5 nM oligomycin A, and assays without bovine heart mitochondria (background) or with only 62.5 nM oligomycin A. (B) Assays of 4 × 105 PMBCs with or without 62.5 nM oligomycin A. The equations of the trendlines are indicated. The slopes of the trendlines (∆OD320nm/min) were used to calculate the oligomycin A-sensitive, reverse CX-V activity relative to the total ATPase activity.
Fig 2.
Reverse CX-V activity in PBMCs cultured in the presence of increasing concentrations (0–100 nM) of oligomycin A as stressor for 3 days.
Shown is the mean percentage of the reverse CX-V activity relative to total ATPase activity in PBMCs cultured for 3 d in the absence of oligomycin A (0 nM) or in the presence of increasing concentrations of oligomycin A (1, 10 and 100 nM) as stressor (n = 6). After culturing, the cells were assayed without and with 62.5 nM oligomycin A to determine the total and oligomycin A sensitive ATPase activity, respectively. The oligomycin A sensitive ATPase activity represents the reverse CX-V activity. Individual data points of the six cultures are shown; data points from corresponding cultures are connected. Statistically significant differences are indicated with their P values.
Fig 3.
∆Ѱm of PMBCs cultured for 3 d in the presence of increasing concentrations (0–100 nM) of oligomycin A as stressor and subsequently analyzed in the absence or presence of the mitochondrial toxins oligomycin A, rotenone+antimycin A or FCCP.
Shown are the individual data points of the ratio (%) of ∆Ѱm from cultures analyzed (A) with and without oligomycin A (n = 4), (B) with and without rotenone+antimycin A (n = 5), or (C) with and without FCCP (n = 6). Data points from corresponding cultures are connected. Statistically significant differences are indicated with their P values.
Fig 4.
Determination of the detection limit of in-gel CX-V activity staining.
(A) In-gel CX-V activity staining of extracts from increasing numbers of PBMCs. (B) Quantification of the in-gel CX-V activity signals in extracts from increasing numbers of PBMCs.
Fig 5.
In-gel CX-V activity and assembled CX-V in PBMCs cultured in the presence of increasing concentrations of oligomycin A for 3 days.
(A) In-gel CX-V activity staining (2.5 × 105 cells) and western blot (2.5 × 104 cells) of clear native gels of a representative PBMC culture probed with an antibody against the ATP5A subunit of CX-V. (B) Quantification of the in-gel CX-V activity signals. (C) Quantification of the assembled CX-V signals on the western blots. Shown is the mean in-gel CX-V activity signal or CX-V assembly signal relative to the signal in cultures not treated for 3 d with oligomycin A (0 nM; n = 6). Individual data points of the six cultures are shown; data points from corresponding cultures are connected. Statistically significant differences are indicated with their P values.