Pp2a clone 1d6

Cell lysates were subjected to immunoblotting using the following antibodies: TY1 (clone MAb-054-050; Diagenode, Denville, NJ, USA), tubulin (clone B-5-1–2; Santa Cruz Biotechnology, Dallas, TX, USA), HA (clone 3F10; Roche, Penzberg, Germany), Akt1 (clone C-20; Santa Cruz Biotechnology), Phospho-Akt (Thr308) (clone D25E6; Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt (Ser473) (clone D9E; Cell Signaling Technology), PP2A (clone 1D6; Millipore, Billerica, MA, USA), Phospho-PP2A alpha (clone E155; Abcam, Cambridge, UK), FLAG (M2 FLAG; Sigma-Aldrich, St. Louis, MO, USA), ERK1/2 (clone C-16/C-14; Santa Cruz Biotechnology), and I2PP2A (SET) (clone E-15; Santa Cruz Biotechnology). Immunoprecipitation was performed in an immunoprecipitation buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 1% Triton, 2 mM sodium orthovanadate, 2 mM PMSF, 50 mM sodium fluoride) as previously reported.^{14 (link)}

Total protein concentration was determined using a bicinchoninic acid (BCA) assay (cat# 23227, BCA Protein Assay Reagent Kit, Thermo Fisher Scientific Pierce Protein Research Products, Rockford, IL). Tissue and cell lysates (20-60 μg total protein) were boiled for 3 min in Laemmli buffer and separated using 7-12% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA), before electrophoretic transfer onto PVDF membranes. The membranes were blocked in 5% nonfat milk and then probed with an antibody (diluted 1:1000) that recognized specifically catalase (D4P7B, Cat# 12980), STAT1 (Cat# 9172) and pSTAT1(Y701) (D4A7 Cat# 7649), FoxO3a (75D8 Cat#2497) and p-FoxO3a(S253) (D18H8 Cat# 13129), Akt and p-Akt(S473) (C67E7 Cat# 4691), PP2A (clone 1D6, Cat# 05-421, Millipore), and GAPDH (Cat# Ab8245), diluted 1:5000. After washes, the blots were incubated for 1 h at room temperature with Alexa Fluor 680- or 790-conjugated AffiniPure secondary antibody (1:10,000 dilution). The bands were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE), and densitometric analysis was performed using Image Studio Software (LI-COR Biosciences, Lincoln, NE).