Ecl chemiluminescence detection reagent

Trilobatin, palmitate, DAPI and (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2NBDG) uptake measurement kits were purchased from Sigma (St. Louis, MO, USA). IRS1, p-IRS1 (Ser 612), p-IRS1 (Ser 307), Akt, p-Akt (Ser 473), p-Akt (thr308), Na, K-ATPase, MYH1, MYOD1, and β-actin primary antibodies were bought from Cell Signaling Technology (Danvers, MA, USA). GLUT4 antibody was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated GAPDH primary antibody was purchased from Aksmics (shanghai, China), Specific anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). Rat/mouse insulin ELISA kits (EZRMI-13K) and ECL chemiluminescence detection reagent were obtained from Millipore (Billerica, MA, USA). Plasma membrane protein extraction kit, nuclear/cytosolic fractionation kit, RIPA buffer and BCA protein assay kit and other chemicals were purchased from Beyotime (Shanghai, China).

Trilobatin, phloridzin, canagliflozin, empagliflozin were purchased from Sigma (Sigma, St. Louis, MO, USA). Cell-light EDU Appollo567 in vitro kit was purchased from RIBOBIO (Guangzhou, China). Rat SGLT2 primary antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). HNF4α and HBXIP primary antibody was from Proteintech (Rosemont, IL, USA). Specific anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). ECL Chemiluminescence Detection Reagent was obtained from Millipore Corporation (Billerica, MA, USA). BCA protein assay kit and other chemicals were purchased from Biyotime (Shanghai, China).

The cells were lysed in RIPA buffer and protein concentration was measured using Bradford’s method. Nuclear proteins were isolated as previously described [18 (link)]. Briefly, the residual pallet was solubilized in 100 µL NLB buffer (50 mM Tris (pH 8.1), 10 mM ETDA, 1% SDS). Afterwards, samples were sonicated (Bioruptor, Diagenode, Seraing, Belgium). Further, 20 µg of total and nucleus protein were separated on 8% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and blotted on nitrocellulose membranes. Protein transfer was validated using Ponceau-S staining. The membranes were blocked with 5% low fat milk for 1 h and incubated with the specific primary antibody at 4 °C overnight (Table 1). A peroxidase conjugated secondary antibody was used to detect the primary antibody and incubated at room temperature for 1 h. Protein bands were visualized with ECL chemiluminescence detection reagent (Millipore, Billerica, MA, USA), which were analyzed densitometrically using ImageJ [19 (link)].