Comprehensive Protein Extraction and Analysis

Cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% NP-40) supplemented with protease inhibitor cocktail. Cell lysates were centrifuged at 12,000 rpm for 30 min at 4 °C, supernatants were saved, and protein concentrations were determined by BCA protein assay (Thermo Scientific, Rockford, Illinois, USA). Equal amounts of total cell lysates or CM of cell culture were subjected to western blot assays as we described previously [41 (link), 42 (link)] to measure protein expression. Antibodies for western blot analyses were from the following sources: NNMT rabbit mAb (E6N2Z, #33361), SIRT1 mouse mAb (1F3, #8469), Hexokinase II rabbit mAb (C64G5, #2867), LDHA rabbit mAb (C4B5, #3582), c-myc rabbit mAb (E5Q6W, #18583), BAX Rabbit mAb (D2E11, #5023), Bim Rabbit mAb (C34C5, #2933), Bcl2 Rabbit mAb (D55G8 #4223), FOXO3a rabbit mAb (D19A7, #12829), p53 Mouse mAb (1C12, #2524), Acetylated-Lysine Antibody (#9441), and β-actin mouse mAb (8H10D10, #3700) (Cell Signaling Technology, Beverly, MA, USA).