Trans blot turbo pvdf transfer kit

Cell lysates prepared with NuPAGE^TM LDS sample buffer (4X) (Invitrogen) were electrophoresed on NuPAGE 4–12% Bis-Tris Gel (Life Technologies), and proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes with Trans-Blot Turbo PVDF Transfer kit (Bio-Rad). PVDF membranes were blocked with 10% skim milk (Sigma-Aldrich) in TBS, followed by incubation with indicated primary and secondary antibodies (GE healthcare). The specific proteins were detected with the Pierce ECL Western Blotting Substrate (Thermo Scientific). ImageJ was used for quantifications of bands on Western blots.
In this study, the following mouse monoclonal primary antibodies (mAbs) were used: V5-tag (#46-0705) (Invitrogen); OPA1 (#612607), Drp1 (#611113), and Myc-tag (#551101) (BD Biosciences); GAPDH (#sc-32233) (Santa Cruz); and Mitofusin 1 (#ab57602) (Abcam). Rabbit polyclonal antibodies (pAbs) were MIEF2 (#HPA042334), Fis1 (#HPA017430) (Atlas Antibodies); MIEF1 [25 (link)]; Mitofusin 2 (#9482S), PARP (#9542) (Cell Signaling); and VDAC1 (#ab15895) (Abcam). The following secondary antibodies were used: The peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies (GE Healthcare) for immunoblotting and the DyLight 488- and 649-conjugated anti-mouse and anti-rabbit IgG antibodies (Vector Laboratories) for immunofluorescence.

Organs were collected from WT, AAVR-KO and SELECTIV-WB mice after euthanasia. Organs were placed into gentleMACS M Tubes (Miltenyi Biotec, 130-093-236) with 3–5 ml of DMEM and lysates were made using gentleMACS Octo Dissociator (Miltenyi Biotec, 130-095-937). Laemmli sample buffer (Bio-Rad, 1610737) supplemented with 2-Mercaptoethanol (Bio-Rad, 1610710) was added to the lysates. Samples were headed at 95 °C for 10 min and cooled on ice before analysis by SDS–PAGE. Samples were run on 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad, 4561086) and transferred using the Trans-Blot Turbo PVDF transfer kit (Bio-Rad, 1704272). Western blotting was performed using the primary antibody rabbit anti-AAVR at a 1:1,000 dilution (Proteintech, 21016-1-AP) and secondary antibody anti-rabbit-HRP at a 1:4,000 dilution (Genetex, GTX213110-01). GAPDH was detected on the same blots using mouse anti-GAPDH-HRP at a 1:4,000 dilution (Genetex, GTX627408-01). Blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34096) or Supersignal West Dura Extended Duration Substrate (Thermo Scientific, 34076). Imaging was performed on a ChemiDoc Imaging System (Bio-Rad) and density was analyzed using Image Lab Software (Bio-Rad).