Anti glut4

Evaluation of protein expression was performed as previously described (11 (link), 28 (link)). Briefly, frozen muscle samples were homogenized in sucrose buffer pH 7.4 (10 mmol/L Tris–HCl, 1 mmol/L EDTA and 250 mmol/L sucrose), centrifuged at 760 g for 10 min at 4°C, and the supernatant was used as a total cellular protein fraction. Protein concentration was determined by Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).
Equal amounts of protein (20–50 μg, according to the target protein) were electrophoresed, transferred to nitrocellulose membrane and immunoblotted with anti-GLUT4 (1:3500, EMD Millipore, Billerica, MA, USA, #07-1404), anti-HK2 (1:1,000, Cell Signaling Technology, Boston, MA, USA, #2867S), anti-NFKB1 (1:1000, Cell Signaling Technology, Boston, MA, USA, #12540S) or anti-RELA (1:450, Abcam, Cambridge, MA, USA, #7970). The membranes were incubated with appropriate secondary conjugated antibody, according to manufacturer's specifications, and signal was detected by enhanced chemiluminescence procedure. The optical density of the blots was quantified by densitometry (ImageScanner III, GE Healthcare, Uppsala, Sweden) and normalized by the densitometry of the respective lane measured in the Ponceau S stained membrane (29 (link)). Results were expressed as arbitrary units (AU) per μg of protein, and considering the mean of control values as 100.

Total cell lysates were prepared by lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, and 1% Triton X-100). On the other hands, membrane fraction was obtained according to previous report^{22 (link)}. In brief, cells were harvested with buffer A (50 mM Tris-HCl, pH 8.0, and 0.5 mM dithiothreitol) containing 0.1% NP-40), and disrupted by passing through a 25-gauge needle 10 times. The homogenate centrifuged at 1,000 × g for 10 min at 4 °C, and the precipitate was suspended in NP-40-free buffer A and recentrifuged at 1,000 × g for 10 min at 4 °C. The precipitate was suspended again in buffer A containing 1.0% (v/v) NP-40 and incubated for 1 h at 4 °C. After the incubation, each homogenate was centrifuged at 16,000 × g for 2 0 min at 4 °C. The supernatant was collected and stored as the plasma membrane fraction at -80 °C until use. Samples were separated by SDS-PAGE, then blotted onto PVDF membrane. Antibodies for the detection of specific protein were anti-AKT (#9272, Cell Signaling Technology), anti-Phospho-AKT (Ser473) (#4058, Cell Signaling Technology), anti-GLUT4 (07–1404, Merck), and anti-Actin (A2066, Sigma-Aldorich). The bands visualized using LuminoGraph I (Atto Co., Tokyo, Japan).

Cellular proteins were collected with SDS-lysis buffer containing protease inhibitor
cocktail (Sigma Aldrich, St. Louis, MO, USA) and benzonase digested. Tissue was
collected in RIPA buffer with PIC. Tissues were homogenated using a Dounce
homogenizer. After centrifugation (14,000 rpm; 15 min; 4°C) and removal of
eventual fat layers, protein concentrations were determined with the bicinchoninic
acid protein assay kit (Pierce, Rockford, IL, USA). Primary antibodies used were
anti-APMAP mouse monoclonal 46F raised against full-length human APMAP (epitope
unknown; Abcam, Cambridge, MA, USA, or Novus Biologicals, Littleton, CO, USA),
anti-GLUT4 (Merck Millipore, Billerica, MA, USA), anti-β-actin
(Sigma-Aldrich), anti-His (GE Healthcare), anti-Flag (Sigma-Aldrich), anti-LOXL3
(Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (Cell Signaling
Technology, Danvers, MA, USA), anti–α tubulin (Abcam), anti-phospho-Akt
(Ser473) XP (Cell Signaling Technology), and anti-Akt (pan) (Cell Signaling
Technology). Secondary antibody signals were visualized by ECL SuperSignal West Pico
Chemiluminescence substrate (Pierce) or Amersham ECL prime substrate (GE Healthcare)
using the G:Box detection system (Syngene, Frederick, MD, USA).