Ripa lysis buffer

Total protein was extracted from cells using RIPA lysis buffer (TaKaRa) according to the manufacturer's instructions. Proteins in lysates were determined using the bicinchoninic acid (BCA) assay and 10% SDS-PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with blocking buffer for 2 h at room temperature and then with the primary antibody anti-HK3 (1:1000, ab1262173, Abcam) overnight at 4 °C. Then, the protein was visualized using ECL plus western blotting detection reagents (Biosciences) and detected with an enhanced chemiluminescence kit.

Cell protein was extracted using RIPA lysis buffer (Takara) containing 1 nM PMSF (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. Then, 20 μg protein per well was electrophoresed in 10% SDS-PAGE gels and transferred onto PVDF membrane (Millipore, Bedford, MA, U.S.A.) and incubated with different primary antibodies, including anti-DEPDC1 (ab197246), anti-GAPDH (ab181603), anti-JNK (ab176645) and anti-p-JNK (ab107407) at 4°C overnight. The membrane was then incubated with HRP-labeled secondary at temperature for 2 h. The bands were detected using an ECL Western blotting kit (Invitrogen) and analyzed on a Gel Doc XR System (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Total protein was extracted from cells using RIPA lysis buffer (TaKaRa) according to the manufacturer's instructions, and the protein concentration was quantified using a bicinchoninic acid protein assay kit. Proteins were separated by 10% SDS/PAGE and then transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with blocking buffer for 2 h at room temperature and then with the primary antibody anti‐PFKFB3 (Abcam, Cambridge, MA, USA) and Blotto overnight at 4 °C. Finally, the protein was visualized using ECL plus western blotting detection reagents (Biosciences, San Jose, CA, USA) and detected with an enhanced chemiluminescence kit.

To validate relationship between AGE-albumin and mitogen-activated protein kinases (MAPK) in skeletal muscle, L6 cells were firstly exposed to 400 ng/ $\mathrm{m}\ell$ sRAGE for 36 hrs with or without 800 μg/ $\mathrm{m}\ell$ AGE-albumin for 48 hrs. Effects of sRAGE treatment to L6 cells in hypoxia and glucose deprivation were confirmed by immunoblotting. L6 lysates were isolated by RIPA lysis buffer (TaKaRa, Tokyo, Japan). Concentration of protein samples was measured using the Bicinchoninic acid (Thermo Scientific, Rockford, IL). Equal amounts of proteins (30 μg protein/lane) were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF). PVDF membrane was incubated with appropriate diluted primary antibodies at 4 °C overnight. The membrane was washed with tris buffered saline with 1% tween 20 (TTBS) three times and incubated with secondary antibodies for 1 hr at room temperature. The primary and secondary antibodies are listed in Table S1. The blotting membrane was developed with enhanced chemiluminescence (ECL) on LAS-4000 (Fuji Film, Tokyo, Japan).

Total protein was extracted from cells using RIPA lysis buffer (TaKaRa) according to the manufacturer’s instructions. Proteins in lysates were determined using the bicinchoninic acid (BCA) assay and 10% SDS-PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with blocking buffer for 2 h at room temperature and then with the primary antibody anti-CCND1 (1:1000, ab16663, Abcam) and and anti-CD31 (PECAM1/CD31; 1:1000, ab28364, Abcam) overnight at 4°C. Then, the protein was visualized using ECL plus western blotting detection reagents (Biosciences) and detected with an enhanced chemiluminescence kit.