Snap i d system

Protein expression of PDH subunits, PDK1, PDK2, PDP1, PDP2, MPC1, and MPC2 was determined using Western immunoblotting following standard procedures. Briefly, 20 μg protein/lane was loaded onto a 10% SDS-PAGE gel and run at 100 V for 2 hours. Protein bands were transferred to a PVDF membrane at 100 V for 1½ hours and membranes blocked with TBS-Tween (TBST) + 0.5% skim milk powder (blocking buffer) using the SNAP i.d. system (Millipore). Membranes were incubated with a primary antibody overnight at 4°C at the following dilutions: PDK1 (Abcam) 1 : 1000; PDK2 (Abcam) 1 : 1000; PDP1 (Abcam) 1 : 250; PDP2 (Abcam) 1 : 250; MPC1 (Cell Signaling) 1 : 1000; MPC2 (Cell Signaling) 1 : 1000; and PDH western blotting cocktail (Abcam), 6 μg/mL). Porin (VDAC, Abcam), 1 : 5000, was used as a loading control. Membranes were then washed and incubated with a secondary antibody (goat anti-rabbit or anti-mouse/HRP, 1 : 2000 (Bio-Rad)), washed, and briefly incubated with ECL chemiluminescent detection reagent (Perkin Elmer) and exposed to film. Band expression was quantified using ImageJ Software (NIH) and normalized to Complex V δ subunit (PDH cocktail) or Porin expression (all other antibodies). Expression of PDK4 was quantified using immunocapture-based ELISA kits (Abcam). PDH enzyme activity was determined spectrophotometrically as described by Pepe et al. [16 (link)].

Extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to a polyvinylidene difluoride membrane (Hybond-P, GE healthcare). Membranes were incubated with the indicated antibodies using the SNAP i.d. system (Millipore). A Sigma anti-FLAG M2 mouse monoclonal antibody or Clontech anti-GFP JL-8 monoclonal antibody was used as the first antibody, and a goat anti-mouse IgG H&L chain-specific peroxidase conjugate (Merck) was used as the second antibody. Immunoreactive bands were detected by a horseradish peroxidase chemiluminescent substrate reagent kit (Novex ECL, Invitrogen) and exposed to FUJI super RX Medical X-ray film (Fujifilm). The uncropped images are shown in Supplementary Figure 12.

T75-cm² flasks of HEK-293 cells were lysed in 1.0 ml of lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 % glycerol, 1 % triton, 1 mM EGTA supplemented with 10 mM N-ethylmaleimide and protease inhibitors). Protein concentration was systematically determined by performing a Bradford assay (Coo protein dosage kit; Interchim, Montluçon, France). Eighty micrograms of proteins were loaded on an SDS-PAGE gel. Protein transfer was done with the dry system transfer iBlot® from Invitrogen (Invitrogen, Basel, Switzerland). Immunoblotting was accomplished by using the SNAP id® system of Millipore (Millipore, Zug, Switzerland). Fluorescent secondary antibodies were used, and detection was realized using the LICOR system® (Lincoln, USA). The intensity of the bands was quantified with the Odyssey software (LICOR).

HEK293 cells or homogenized whole mouse heart were lysed in 1.0 mL of lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 10% glycerol, 1% triton, mM EGTA supplemented with 10 mM N-ethyl maleimide and protease inhibitors (Roche, Basel, Switzerland). Protein concentrations were determined by performing Bradford assays (Coo protein dosage kit; Interchim, Montluçon, France). Forty μg of protein was loaded onto a SDS-PAGE gel. Protein transfer was performed with the dry system transfer i-blot from Invitrogen (Invitrogen, Basel, Switzerland), and immunoblotting using the snap-id system of Millipore (Millipore, Zug, Switzerland). Detection was achieved using the LICOR system^®, and the intensity of the bands was quantified with Odyssey software (LICOR, Lincoln, NE, USA).

The western blot analysis for the HTT protein (17/68Q tract) was performed as previously described (Fiszer et al., 2011 (link)). Briefly, 30 μg of total protein was run on a Tris-acetate sodium dodecyl sulfate (SDS)-polyacrylamide gel (1.5 cm, 4% stacking gel/4.5 cm, 5% resolving gel, acrylamide:bis-acrylamide ratio of 49:1) in XT Tricine buffer (Bio-Rad, Hercules, CA, USA) at 130 V in an ice-water bath. Subsequently, the proteins were wet-transferred to a nitrocellulose membrane (Sigma–Aldrich). All of the immunodetection steps were performed using the SNAPid system (Millipore). The primary antibodies anti-huntingtin (1:1000, MAB2166, Millipore) and anti-plectin (1:1000, ab83497, Abcam, Cambridge, UK) and secondary antibodies anti-mouse HRP-conjugate (1:2000, A9917, Sigma–Aldrich) and anti-rabbit HRP-conjugate (1:2000, 711-035-152, Jackson ImmunoResearch) were used in a PBS/0.1% Tween-20 buffer containing 0.25% non-fat milk. The immunoreaction was detected using WesternBright Quantum HRP Substrate (Advansta, Menlo Park, CA, USA). The protein bands were scanned directly from the membrane using a camera and were quantified using Gel-Pro Analyzer.