Odyssey m imaging system

Worms grown to day 1 of adulthood were washed off the plate using M9, harvested, and flash-frozen in liquid nitrogen. Then, animals were suspended in lysis buffer [100 mM Hepes (pH 7.4), 300 mM NaCl, 2 mM EDTA, and 2% Triton X-100, with EDTA-free protease inhibitor cocktail (Roche)]. Next, animals were homogenized using the Precellys Tissue Homogenizer, with glass and zirconium beads (2 mm). Lysates were centrifuged (8000g for 5 min at 4°C), supernatant was removed, and protein was quantified using a BCA Protein Assay kit (Thermo Fisher Scientific, 23225). Protein samples were applied on to cellulose acetate membrane with 0.22-mm pore size (Schlechtes + Schule) and assembled in vacuum slot blotter (Bio-Dot, Bio-Rad). Membrane was washed five times with 0.2% SDS on the blotter and subjected to antibody incubation for detecting aggregated protein retained on the membrane. Membranes were incubated with anti-GFP antibody (1:1000 dilution in LI-COR blocking buffer) overnight in a cold room. Membrane was washed three times with tris-buffered saline + tween (TBST) and then incubated with secondary antibody (1:10,000 dilution in LI-COR blocking buffer). Membranes were washed three times with TBST and imaged using the Odyssey M Imaging System (LI-COR) to visualize the protein bands. Bands were then quantified using Fiji.

Worms grown to day 1 of adulthood were washed off the plate using M9, harvested, and flash-frozen in liquid nitrogen. Then, animals were suspended in lysis buffer [100 mM Hepes (pH 7.4), 300 mM NaCl, 2 mM EDTA, and 2% Triton X-100, with EDTA-free protease inhibitor cocktail (Roche)]. Next, animals were homogenized using the Precellys Tissue Homogenizer, with glass and zirconium beads (2 mm). Lysates were centrifuged (8000g for 5 min at 4°C), supernatant was removed, and protein was quantified using a BCA Protein Assay kit (Thermo Fisher Scientific, 23225). Protein samples were applied on to cellulose acetate membrane with 0.22-mm pore size (Schlechtes + Schule) and assembled in vacuum slot blotter (Bio-Dot, Bio-Rad). Membrane was washed five times with 0.2% SDS on the blotter and subjected to antibody incubation for detecting aggregated protein retained on the membrane. Membranes were incubated with anti-GFP antibody (1:1000 dilution in LI-COR blocking buffer) overnight in a cold room. Membrane was washed three times with tris-buffered saline + tween (TBST) and then incubated with secondary antibody (1:10,000 dilution in LI-COR blocking buffer). Membranes were washed three times with TBST and imaged using the Odyssey M Imaging System (LI-COR) to visualize the protein bands. Bands were then quantified using Fiji.

Western blot analysis was carried out as described before [5 (link)]. Briefly, the OSA cells and fibroblasts were lysed with CelLytic M lysis buffer (C2978, Sigma-Aldrich, St. Louis, MO, USA) in the presence of protease inhibitor (P8340, Sigma-Aldrich) and phosphatase cocktail inhibitor B (sc-45045, Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were resolved on Bolt Bis-Tris 4–12% polyacrylamide gels (ThermoFisher Scientific, Waltham, MA, USA) and transferred to polyvinylidene difluoride membranes, incubated with 5% bovine serum albumin (BSA) for 2 h at room temperature, before being incubated with the primary antibodies at 4 °C overnight. The primary antibodies used were PTEN (Cell Signaling Technology, D 4.3, 1:2000), p16^INK4A (Santa Cruz Biotechnology, F-8, 1:500) and β-actin (Cell Signaling Technology, 8H10D10, 1:2000. The secondary antibodies were obtained from (LI-COR Biosciences, donkey anti-mouse; goat anti-rabbit (LI-COR Biosciences), both used at a 1:15,000 dilution and incubated with the blot for 1 h at room temperature. The membranes were visualized using the Odyssey^® M Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using Image Studio™ Lite software 5.2.5 (LI-COR Biosciences, Lincoln, NE, USA).

Unless specified otherwise, samples were heated in Laemmli buffer (from Bio-rad, no. 1610747) for 5 min at 95 °C, with or without 50 mM dithiothreitol (DTT; from GoldBio), and electrophoretically resolved under denaturing conditions on 4–20 % TGX precast polyacrylamide gels (from Bio-Rad). For Coomassie-blue staining, gels were first fixed in 50 % v/v methanol and 10 % v/v acetic acid for 30 min at RT, then incubated for a further 30 min in 50 % v/v methanol, 10 % v/v acetic acid, and 0.001 % w/v Coomassie Brilliant Blue R-250 (from BioRad), and finally destained overnight in 10 % v/v acetic acid before being washed into water. For immunoblotting, proteins were transferred overnight onto nitrocellulose membranes (from Bio-Rad, no. 1620112). Membranes were blotted in TBST buffer (20 mM Tris•NaOH pH 7.5, 150 mM NaCl, and 0.1 % v/v Tween 20) containing 3 % w/v non-fat milk solids (from Apex, no. 20241) using antibodies against the FLAG epitope (no. F7425) or the SBP tag (no. MAB10764), and fluorescently-labeled secondary antibodies IRDye 800CW or IRDye 680CW (from LI-COR Biosciences), as appropriate. Blots were imaged on a LI-COR Odyssey M imaging system.