Femto chemiluminescence substrate

Immunoblotting assays for whole cell lysates of cells and tissues were performed as previously described (Tarrago et al., 2018 (link)). Tissues or cells were homogenized and lysed in NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40) supplemented with 50 mM β-glycerophosphate, 5 mM NaF and a protease inhibitor cocktail (Roche). After 30 min of incubation at 4°C, the samples were centrifuged at 12,000 r.p.m. for 10 min at 4°C. Protein concentrations in the supernatants were determined by Bio-Rad protein assay. Lysates were separated by SDS–PAGE, and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). Enhanced chemiluminescence detection was performed using Super-Signal West Pico or Femto Chemiluminescence Substrate (Thermo Scientific). Films were scanned and densitometry was performed using ImageJ. The following antibodies and their dilutions were used for immunoblotting: mouse CD38 (R&D Systems; AF4947, 1:1,000), CD45 (Abcam; AB40763; 1:1000), Phospholamban (PLN) (Invitrogen; MA3-922; 1:1000), CYP3a4 (Proteintech, 18227-1-AP; 1:1000), actin (Cell Signaling Technology; 8457, 1:5,000) and GAPDH (Cell Signaling Technology; 97166, 1:5,000).

Tissue extracts were homogenized using a Bead Mill 24 homogenizer (Waltham) and lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), 5 mM NaF, a protease inhibitor cocktail (Millipore Sigma), and 5 mM nicotinamide. After incubating 30 min at 4 °C, samples were centrifuged at 15,000 rpm for 10 min at 4 °C. Protein concentrations of the supernatants were determined by the Bradford protein assay (BioRad). Lysates were separated by SDS-PAGE and transferred by electrophoresis to PVDF membranes (Millipore Sigma). Membranes were immunoblotted with primary mouse α-Klotho antibody (R&D Systems, AF1819) at 1:1000 dilution and GAPDH antibody (Cell Signaling Technology, 97166) at 1:1000 dilution. Enhanced chemiluminescence detection was performed using SuperSignal West Pico or Femto Chemiluminescence Substrate (Thermo Scientific). All blots were imaged with a GelDoc Go Imaging System (BioRad).