Anti cox4

To determine the expression of mitochondria-related genes in TIMP-3 KO mice, soleus muscle was harvested and homogenized. Ten µg of protein from each sample was denatured with 4X Sample Buffer Solution with 3-mercapto-1,2-propanediol (Wako, Osaka, Japan) and subjected to SDS-PAGE. Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane followed by blocking with PVDF Blocking Reagent for Can Get Signal (Toyobo, Osaka, Japan) for 1 h at room temperature. After blocking, membranes were probed with primary antibodies for 1 h at room temperature, secondary antibody for 1 h at room temperature, and visualized with the ECL detection system. The intensity of each band was quantified using NIH ImageJ analysis software v1.61 (National Institutes of Health, Bethesda, MD) and normalized to GAPDH signal. The antibodies used were anti-UCP-2 (1∶1000; Proteintech, Chicago, IL, USA), anti-NRF-1 (1∶1000; abcam, Cambridge, UK), anti-NRF-2 (1∶500; abcam, Cambridge, UK), anti-VDAC (1∶1000; Cell Signaling Technology, Danvers, MA, USA), anti-COX4 (1∶1000; GeneTex, Irvine, CA, USA) and anti-GAPDH (1∶30000; Cell Signaling Technology, Danvers, MA, USA).

After treatments, cells were fixed with acetone/methanol (1:1) for 5 min at −20 °C and blocking in 5% BSA in PBS for 30 min. Fixed cells were incubated overnight at 4°C with primary antibody [anti-SDHA (1:500, mouse, Invitrogen), anti-PDI (1:500, rabbit, Stressgen), anti-ATF4 (1:500, rabbit, Santa Cruz Biotechnologies), anti-CHOP (1:500, rabbit, Cell Signaling), anti-cytochrome c (1:500, mouse, BD Transduction Lab.), or anti-COX IV (1:500, mouse, GeneTex)]diluted in PBS and then washed three times in PBS and incubated for 1 h at room temperature with anti-rabbit or anti-mouse Alexa Fluor 488 or 594 (1:1000, Molecular Probes). Slides were mounted with ProLong Gold antifade mounting reagent (Molecular probes) and cell staining was visualized with a fluorescence microscope using Zeiss filter sets #46 and #64HE.