Nupage sds page gel system

The commercial NuPAGE SDS-PAGE gel system (Life Technologies) was used for protein separation and blotting. Proteins were separated using 4−12% Bis-Tris gels in NuPAGE running buffer according to the manufacturer's instructions. Proteins were blotted onto PVDF membranes (Millipore) using the described standard conditions. Blots were incubated in PBS containing 0.1% Tween-20 (PBST) for 15 min, stained with Ponceau-S for 5 min and imaged on an LAS-4000 mini (Fujifilm). The blots were then washed in PBST for 30 min with several rounds of buffer exchange and gentle agitation.

For cellular lysis, cells were washed twice in phosphate buffered saline and lysed in protein lysis buffer. Lysates were incubated on ice for 30 min and subsequently centrifuged at 4°C and 13000 rpm for 15 min. Protein concentration of the supernatant has been determined using a Bradford protein assay (Bio-Rad). After addition of sample buffer, dithiothreitol, and H₂O, the protein has been denatured at 95°C for 5 min. Protein mix was run on commercial precast 4–12% Bis-Tris gels (NuPAGE^® SDS-PAGE Gel System, LifeTechnologies, Carlsbad, CA, United States) and transferred onto a nitrocellulose membrane by a commercial wet/tank blotting system (Trans-Blot^®, Bio-Rad Laboratories, Hercules, CA, United States). After blocking with 5% milk, the blots were incubated with the primary antibody (anti-SGLT2, ab37296, Abcam or anti-GAPDH, sc-32233, Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C. The proteins were then detected by enhanced chemiluminescence (Pierce ECL Plus, Thermo Scientific) after labeling with a horseradish peroxidase-labeled secondary antibody according to the manufacturer’s instructions (sc-2056, sc-2314, or sc-2004, Santa Cruz). Densitometric analysis was performed with ImageJ 1.49v.

Lysates for western blotting were prepared using standard lysis buffer (50 mM Tris HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100) containing protease inhibitor (p8340, Sigma) and 0.2 mM sodium orthovanadate. The NuPAGE SDS PAGE Gel System and NuPAGE Bis Tris Precast Gels (4–12% and 12%) (Life Technologies) were used to perform gel electrophoresis. Western Lightning PLUS Enhanced Chemiluminescent Substrate (PerkinElmer) was used for imaging western blots on the Vilber Lourmat Fusion chemiluminescent imaging system. Quantitative western blotting was performed using multistrip western blotting, as performed previously (Kennedy et al., 2019 (link)).

Lysates for western blotting and immunoprecipitation were prepared using the normal lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100) containing protease inhibitor cocktail (p8340, Sigma) and 0.2 mM sodium orthovanadate. Immunoprecipitation was performed using protein-A/G agarose beads (Invitrogen), as previously described [27 (link)]. SDS-PAGE electrophoresis and western blotting were performed using the NuPAGE SDS PAGE Gel System and NuPAGE Bis Tris Precast Gels (4–12%) (Life Technologies). Western Lightning PLUS Enhanced Chemiluminescent Substrate (PerkinElmer) was for imaging western blots on the Vilber Lourmat Fusion chemiluminescent imaging system. Quantitative western blotting was performed using multistrip western blotting [28 (link)]. The Human Phospho-Kinase Antibody Array was obtained from R&D Systems (MN, USA) and used according to manufacturer’s instructions.

Routinely, Ldi expression levels in E. coli cell lysate and the levels of purified Ldi were assessed by SDS-PAGE or Western blot. SDS-PAGE was performed with the NuPAGE^® SDS-PAGE Gel System (Life Technologies, Austria) according to the manufacturer’s recommendations. For analysis of proteins via immunoblot, a primary anti-His antibody as well as an HRP-conjugated secondary antibody and enhanced chemiluminescent signal detection (SuperSignal^TM, Pierce Biotechnology, Rockford, IL, USA) were used for visualization of immunoreactive bands.

Western blotting was conducted using NuPAGE SDS-PAGE Gel System (Life Technologies). Cell lysates were prepared in NETN 150 buffer (sodium chloride, 150 mM; EDTA, 2 mM; Tri-HCl, 50 mM; NP-40, 1%) containing protease inhibitor cocktail (cOmplete mini, Roche). Protein separation and Western blotting were performed according to the manufacturer’s instructions^{69 (link),70 (link)}.