Anti ampkα1

Primary MEFs were cultured as described above and plated in 2 × 15 cm tissue culture dishes and incubated overnight under normal growth conditions. 24 h after plating, cells were treated with 20 μM MG132 or DMSO for 2.5 h prior to harvest. Cells were washed 1X in cold PBS and lysed in Cell Lytic M (Sigma) containing 1X HALT protease/phosphatase inhibitor (Pierce) and 50 μM PR619 (LifeSensors). Lysates were clarified by centrifugation at 15,000 × g for 10 m. Total protein concentration was determined by BCA protein assay (Pierce) and 1.5 mg total protein clarified lysates were incubated overnight at 4 °C with 10 μg of anti-AMPKα1 (R and D Systems, AF3197), anti-CHIP (Abcam, ab4447), rabbit IgG or goat IgG antibodies. 120 μl Protein-G Dynabeads (Invitrogen) was then added to each sample and incubated for 0.5 h at room temperature with rotation. Beads were washed four times with Phosphate-Buffered Saline with 0.05% Tween-20; subsequently, proteins were eluted in SDS-sample buffer and analyzed by SDS-PAGE and western blotting using anti-CHIP (Sigma, S1073), anti-AMPKα1/2 (Cell Signaling Technologies, 2532) or anti-Hsc70 (Enzo, ADI-SPA 815) antibodies.

Antibodies directed against AMPKα1/α2, ACC, pSer⁷⁹-ACC were obtained from Cell Signaling Technology. Anti-AMPK α1 and anti-AMPK α2 antibodies were from R&D Systems. Anti p-Thr¹⁷²AMPK antibody and p-Thr¹⁷²AMPK blocking peptide were obtained from Santa Cruz Biotechnology24 (link). Anti-actin antibody was from BD Transduction Laboratory. Anti-MAP2, anti-α-tubulin and anti-acetylated-α-tubulin antibodies were from Sigma. Mouse monoclonal antibodies PHF1 (tau pSer³⁹⁶/Ser⁴⁰⁴)50 (link), CP13 (tau pSer²⁰²)51 (link), 2E12 or RZ3 (tau pThr²³¹)5 (link), DA9 (total tau aa102-140)36 (link), DA31 (total tau aa220-240)36 (link) and MC1 (conformation dependent antibody that recognizes only tau in a pathological conformation)52 (link) were previously described. 12E8 monoclonal antibody was obtained from Dr. Seubert (Elan Pharmaceuticals, San Francisco, CA; tau pSer^262/356)53 (link). As total tau antibody in the Western-blot experiments, we used tau Cter (homemade well characterized antibody recognizing the 11 amino-acids in the c-terminal part of tau27)54 (link). Tau5 (total tau) and AT180 (tau pThr231) were from Invitrogen.
AICAR, metformin, PD98059, H-89, LY294002 were purchased from Tocris; roscovitin and MARK/Par1 inhibitor from Merck; Compound C was from Santa-Cruz Biotechnology and rapamycin was from Cell Signaling Technology.

20 μg of cells or 40 μg of tissue lysates were subjected to electrophoresis on 10% acrylamide gels and transferred to PVDF membranes. The membranes were incubated for 1 h with blocking buffer (either TBS-T containing 5% [w/v] BSA or 5% skim milk). The membranes were then incubated with the indicated primary antibodies diluted in blocking buffer (1:1,000) for 12 h at 4 °C: anti-pAmpk-α Thr172 (Cell Signaling, #2535s), anti-Ampk-α1 (R&D, #AF3197), anti-Ampk-α (Cell Signaling, #2532), anti-Ampk-α2 (abcam, ab3760), anti-UCP1 (abcam, ab10983), anti-HSP90 (Cell Signaling, #4874), anti-Bcl2 (abcam, ab196495), anti-cleaved Caspase 3 (Cell Signaling, #9661), anti-GAPDH (Cell Signaling, #2118), anti-Reg3γ (abcam, ab198216), and anti-Reg3α (abcam, ab202057). The membranes were washed three times with TBS-T and incubated with the secondary HRP-conjugated antibodies mouse anti-rabbit IgG-HRP (Cell Signaling, #7074S) (diluted 1:2000 in 5% skim milk) at room temperature for 1 h. Finally, the membranes were washed in TBS-T three times for 10 min each, and the signal was detected using enhanced chemiluminescence reagent (Pierce, IL, USA). Protein levels were quantified by densitometry using Image J software.