Anti phosphorylated ampkα thr172 antibody

Samples were subjected to electrophoresis, Western blot, and antibodies incubation using the methods published previously [7 (link)]. Briefly, 200 μg of kidney cortex were loaded on a 6–10% polyacrylamide gel and separated by electrophoresis (200 volts, 90 min). Following transfer to a nitrocellulose membrane (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA), the membranes were incubated with Ponceau S red (PonS) stain solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 min on the rocker to verify equal loading. After blocking with phosphate-buffered saline–Tween (PBS-T) containing 5% dry milk, the membranes were incubated with primary antibody. The blots were incubated overnight at 4 °C with a 1:1000 dilution of anti-phosphorylated AMPKα (Thr172) antibody (1:1000, #2535, Cell Signaling, Danvers, MA, USA). Following five washes with 0.1% Tween-Tris-buffered saline (TBS-T), the membranes were incubated for 1 h with horseradish peroxidase-labeled secondary antibody diluted 1:1000 in TBS-T. Bands were visualized using SuperSignal West Pico reagent (Pierce; Rockford, IL, USA) and quantified by densitometry as integrated optical density (IOD). IOD was then normalized to total protein PonS staining. Protein abundance was expressed as IOD/PonS.

Lysophosphatidic acid (LPA) (oleoyl C: 18:1) was obtained from Avanti Polar Lipids (Alabaster, AL, United States). 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5-isoxazolyl]benzylsulfanyl)propanoic acid (Kil6425) was purchased from Sigma (St. Louis, MO, United States). PI3K inhibitor LY294002, rapamycin, anti-phosphorylated mTOR (ser2448) antibody, anti-total mTOR antibody, anti-p70s6 kinase (Thr389) antibody, anti-phosphorylated p70s6 kinase, anti-4E-BP1 antibody, anti-phosphorylated 4E-BP1(Thr37/46) antibody, and anti-phosphorylated AMPKα (Thr172) antibody were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-p62/SQSTM1 (sequestosome1) polyclonal antibody and anti-beclin1 polyclonal antibody were from Proteintech Technology (Proteintech Group, Wuhan, China), and anti-LC3B polyclonal antibody and anti-GAPDH monoclonal antibody were obtained from Sigma (St. Louis, MO, United States). Lipofectamine^TM RNAiMAX, stealth siRNA, and siRNA negative control were purchased from life Technologies (Invitrogen, Carlsbad, CA, United States). The PowerUp^TM SYBR Green Master Mix assay (Applied Biosystems, Life Technologies, Foster City, CA, United States).