Mini protean tgxtm precast protein gels

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis was conducted as previously described in Bågenholm et al. [18 (link)], using either an unstained PageRuler protein ladder (for RhMan113A and RhGal36A) (Thermo Scientific, Waltham, MA, U.S, product number: 26614) or a pre-stained PageRuler (RhMOP130A) (26616, Thermo Scientific, Waltham, MA, USA) as reference. Electrophoresis was carried out by using 12% Mini-PROTEAN^® TGXTM Precast Protein gels (4561046, Bio-Rad, CA, USA) for 50 min at 150 V. Native polyacrylamide gel electrophoresis (native-PAGE) was conducted with Native PAGE marker (Thermo Fischer, Waltham, MA, USA, product number LC0725). Protein samples were loaded onto 4–15% Mini-PROTEAN^® TGXTM Precast Protein Gels (4561086, Bio-Rad, CA, USA), and the electrophoresis was run for 30 min at 150 V, using the manufacturer’s instructions.

The nuclear and cytoplasm lysate samples were denatured at 95°C for 5minutes in 1x Laemmli buffer prior to loading onto a 4–15% SDS-PAGE (4–15% Mini-PROTEAN® TGX^TM Precast Protein Gels, Bio-Rad #4561085). 10uL Precision Plus Protein^TM Kaleidoscope^TM Prestained Protein Standards (Bio-Rad, #1610375) or 10-15uL of samples were loaded on to the gel. The gel was eletrophorised for 35-40mins at 200mV, in 1x running buffer (25mM Tris-Base, 190mM glycine, 0.1% SDS, pH 8.3). Proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat no. 162–0115) at 400mA, 300V for 1 hour and 30 mins at 4°C, in 1x transfer buffer (25mM Tris-Base, 190mM glycine, 20% methanol, pH 8.3). Primary antibodies (anti-FLAG 1:1000, Sigma Cat no. F1804-200UG, or anti-GAPDH1:1000, Millipore Cat no MAB374) were diluted in 1% skim milk in PBS and incubated for 1 hour at RT with shaking. After incubation, membranes were washed with 1xPBS+0.1%Tween for three times 5 mins and once for 10 mins. After washing, membranes were incubated with secondary antibody (1:5000 goat anti- mouse Ig-HRP, Sigma Cat no. A44161ML) diluted in 1% skim milk in PBS, for 1 hour at RT with shaking, and then washed again as above. Membrane was visualised under chemiluminescence with HRP substrate (Millipore, Cat no. WBLUF0500) at various exposure times.

The LRP2 siRNA- or scRNA-transfected C₂C₁₂-myc-GLUT4 cells were serum starved for 4 h, followed by the stimulation with 100 nM insulin for 0–30 min. The cells were rinsed with ice-cold PBS, followed by 0.3 mg/mL sulfo-NHS-Biotin (Thermo fisher scientific) labeling at 4 °C for 30 min. Then, the cells were quenched with ice-cold 100 mM glycine at 4 °C for 10 min. The cells were washed three times with ice-cold PBS, and then lysed with NP40 Cell Lysis Buffer (Thermo fisher scientific) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo fisher scientific) and 1 mM PMSF. The 100 μg total protein was incubated with 10 μl streptavidin-agarose beads (50% slurry, Thermo fisher scientific) at 4 °C overnight to pull down the biotinylated surface proteins. The beads were washed with lysis buffer three times, and then boiled in SDS–PAGE loading buffer (NuPAGE™ LDS Sample Buffer containing with NuPAGE™ Sample Reducing Agent (Thermo fisher scientific) at 100 °C for 10 min. The biotinylated surface protein fraction and the total protein lysate were resolved on 4–16% Mini-PROTEAN TGX^TM precast protein gels (Bio-Rad, Hercules, California), transferred to nitrocellulose membrane, and immunoblotted using each antibody. The bands were visualized with enhanced chemiluminescence and quantified by an ImageJ program.