Mitoprofile total oxphos antibody cocktail

Cell lysates were prepared and immunodetection was performed as described previously^{15 (link)} using MitoProfile total OXPHOS antibody cocktail (Mitosciences, Eugene OR) anti-Mn-SOD (EMD Millipore Corporation, Billerica MA) and anti-GAPDH (Cell Signalling Technology, Danvers MA, USA) as loading control. Densitometry was performed using the gel-scan program QuantityOne.

THP-1 cells were lysed using a lysis buffer containing 1% Triton X and the protease inhibitor phenylmethanesulfonyl fluoride (1 mM) (both Sigma-Aldrich) and the protein concentration in the lysates determined by Bradford assay. Equal amounts of protein were separated on the basis of size by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes and blotted with different antibodies, assessing the signal intensity after addition of an enhanced chemiluminescent substrate using the MultiSpectral Imaging System (UVP, Upland, CA, USA). The following antibodies were used; anti-mouse Ig-HRP (0260) and anti-rabbit-HRP (0448) from Dako (Cambridge, UK), β-actin (mouse, ab8226) and SOD2 (rabbit, ab13533) from Abcam (Danvers, MA, USA), LC3-I/II (rabbit, CS54995) from Cell Signalling (Beverley, MA, USA), Mitoprofile® Total OXPHOS antibody cocktail (mouse, MS604) from MitoSciences (Eugene, OR, USA) and TFAM (mouse, NBP1-71648) from Novus Biological (Cambridge, UK).

A volume of 20 μg of isolated mitochondria were resuspended in 4 × Lämmli-Buffer (4% SDS, 20% Glycerol, 120 mM Tris, 0,02% Bromophenol Blue), proteins were separated on 4–12% NuPage gels (Invitrogen) and transferred on Hybond^TM-P membrane (GE Helthcare). Primary antibodies used for western blotting were as follows: MitoProfile total OXPHOS antibody cocktail (MitoSciences), HSP60 (Cell signalling, 1:1000), COX1 (Invitrogen, 1:1000), MTERF1 (Proteintech, 1:1000), TFAM (Abonva, 1:1000), SDH2 (MitoScience). Rabbit polyclonal antisera against MGME1, TWINKLE, POLRMT, COX2, LRPPRC proteins were generated using recombinant mouse proteins.
For BN-PAGE 75 µg mitochondria were solubilized in solubilization buffer: 1% (w/v) digitonin (Calbiochem), 20 mM Tris, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% (v/v) glycerol. Following 15 min of incubation on ice, non-solubilized material was removed by centrifugation and the supernatant was mixed with loading dye (5% (w/v) Coomassie Brilliant Blue G-250 (Serva), 100 mM Tris, pH 7, 500 mM 6-aminocaproic acid). Samples were resolved on 3–13% (w/v) acrylamide gradient BN-PAGE gels. BN gels were further subjected to Coomassie Brilliant Blue R staining, in-gel activity assay^{58 (link)} or western blot analysis, as indicated.

Protein expression was analyzed by western blotting using the following antibodies: anti-Drosha, Cell Signaling, dilution 1:1000; anti-p70S6K, Cell Signaling, dilution 1:1000; anti-p-p70S6K (Thr389), Cell Signaling, dilution 1:1000; anti-mTOR, Cell Signaling, dilution 1:1000; anti-p-mTOR (Ser2448), Cell Signaling, dilution 1:1000; anti-β-Actin, Sigma-Aldrich, dilution 1:5000; anti-LC3, Novus Biological, dilution 1:200; MitoProfile Total OXPHOS Antibody Cocktail, MitoSciences, dilution 1:1000. An equal amount of total proteins (12–20 µg) extracted from primary rat hepatocytes were separated by electrophoresis and transferred to polyvinylidene difluoride membranes (Membrane PVDF 0.45 µm, Immobilon-P). Primary antibody incubation was performed overnight at 4 °C and then detected with HRP-conjugated anti-rabbit or anti-mouse immunoglobulins (Vector Laboratories). Immunoreactive proteins were revealed by enhanced chemiluminescence (Luminata Crescendo WB HRP Substrate, Millipore) and the intensity of each band quantified with ImageJ software (US National Institutes of Health, Bethesda, Maryland, USA).