Anti glut4

Protein isolation and immunoblotting procedures were as described previously [22 (link)]. For quantification of sAPP fragments, differentiated cells were treated in 10 ml Optimem (Gibco Life Technologies, Paisley, UK), and media were concentrated (30 kDa Amicon Ultra 15 ml filter) by centrifugation (4,000 g) and subjected to SDS-PAGE with amounts presented relative to total protein. Primary antibodies used were: anti-sAPPβ (Covance, Alnwick, UK; 1:1,000), anti-sAPPα (IBL International, Hamburg, Germany; 1:50), anti-APP (Ab54, GlaxoSmithKline; 1:4,000), anti-phospho-PKB (protein kinase B; Ser⁴⁷³), anti-PKB and anti-HKII (hexokinase II) (Cell Signaling, Hitchin, UK; 1:1,000), anti-GLUT1 (Millipore, Nottingham, UK; 1:1,000) and anti-GLUT4 (Abcam, Cambridge, UK; 1:1,000). Glut1 (also known as Slc2a1), Glut4 (also known as Slc2a4) and HkII (also known as Hk2) mRNA was determined by TaqMan RT-PCR (Applied Biosystems, Paisley, UK; Prism Model 7700) using commercial primers and probe sets.

Protein concentrations of cells and tissue were measured with the Protein Quantitative Kit (Applygen Technologies Inc.). Protein samples were separated by 10% SDS/PAGE gel and transferred onto a poly(vinylidene difluoride) membrane (Merck Millipore, Bedford, MA, USA) by a semi‐dry transfer (Bio‐Rad, Hercules, CA, USA). After blocking with 5% milk, the membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies included: anti‐phospho (p)‐PI3Kp85 (1 : 2000, ab182651; Abcam, Cambridge, UK), anti‐p‐Tyr612‐IRS1 (1 : 2000; Thermo Fisher Scientific), anti‐p‐AKT (Ser473) (1 : 2000; CST, 4060 Cell Signaling Technology, Danvers, MA, USA), anti‐AKT (1 : 1000; CST, 4691 Cell Signaling Technology), and anti‐GLUT4 (1 : 1000; ab654 Abcam). After washing, the membrane was incubated with the corresponding peroxidase‐labeled secondary antibodies. Proteins were visualized using a chemiluminescence kit. (ZSGB‐BIO, Beijing, China). β‐Actin was used as an internal reference. The expressed proteins were quantified by densitometry analysis using imagej software (National Institutes of Health, Bethesda, MD, USA).

Anti-Sx4 (#110042), anti-Sx2 (#110022), anti-Sx3 (#110032), anti-Sx6 (#110062) and anti-Sx16 (#110161) were from Synaptic Systems (Göttingen, Germany) and were all used at 1:1000. Anti-GLUT1 (#652) and anti-GLUT4 (#654) were from Abcam (Cambridge, UK) (both used at 1:1000) and anti-GAPDH (#4300; used at 1:80,000) was from Ambion (Foster City, CA, USA). Detection antibodies were from LI-COR Biosciences (Lincoln, NE, USA) and were used at 1:10,000. Anti-sortilin (#12369-1-AP; 1:1000), anti-tankyrase (#18030-1-AP; 1:250) and anti-USP25 (#12199-1-AP; 1:1000) were from Proteintech. Anti-IRAP was from Paul Pilch (Boston College, Boston, MA, USA; 1:500). Anti-ACRP30 was as described previously (Clarke et al., 2006 (link); 1:1000).

Total protein was extracted from MIN6 cells with cold RIPA lysis buffer containing proteases and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electrotransfer onto a PVDF membrane (Millipore, Billerica, MA, United States) and immunoblotting. Then, the membrane was blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h and incubated overnight at 4°C with anti-Glut4, anti-INS-R, anti-IRS-1, anti-IRS-2, and anti-GAPDH primary antibodies (1:1,000; Abcam, Cambridge, MA, United States). The membranes were washed three times in Tris-buffered saline and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; Gaithersburg, MD, United States) for 1 h. The expression of the specific protein was visualized using a commercial electrochemiluminescence kit (Invitrogen).
Ultra-performance liquid chromatography was coupled with time-of-flight mass spectrometry analysis (UPCL-TOF/MS^E).