Complete proteinase inhibitor cocktail

mESCs were either cultured under serum/LIF conditions or without LIF for 72 h. 2 × 10⁷ cells were harvested, washed twice in ice-cold 1xPBS and lysed in 1 ml of nuclear lysis buffer (NLB: 10 mM Hepes KOH pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.1% NP-40 supplemented with cOmplete™ proteinase inhibitor cocktail (Roche)). Lysate was incubated for 10 min on ice, and nuclei were pelleted at 2000 rpm for 10 min. Nuclear pellet was washed in NLB and lysed in 0.5 ml 1 × RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) Supplementaled with cOmplete™ proteinase inhibitor cocktail (Roche)). The lysate was incubated on ice for 20 min with vortexing every 5 min and centrifuged at full speed for 15 min to remove cellular debris. 2 μg of HP1γ antibody (Millipore 05-690 MAB clone 42s2) or an isotype control (Abcam, HA.C5, ab18181) was added and incubated at 4 °C overnight on a rotating wheel. 30 μl of Protein G Dynabeads^® (Thermo Fisher Scientific) was added and incubated for 2 h at 4 °C on a rotating wheel. Beads were washed 3 ×  in ice-cold 1× RIPA, lysed in 2 × Laemmli buffer, resolved by SDS-PAGE and subjected to immunoblotting.

Retina or cell lysates were prepared by incubating tissue or cell pellets with ice-cold 1x lysis buffer (Beyotime Biotechnology, China) supplemented with cOmplete^TM proteinase inhibitor cocktail (Roche, Switzerland) and phosphatase inhibitor cocktail (Roche, Switzerland) on ice for 20 min. Lysates were then centrifuged at 12,000 g at 4°C for 15 min to remove the insoluble fractions. The supernatant was then mixed with ×5 sample loading buffer (Beyotime Biotechnology, China) and boiled for 7 min. Denatured proteins were separated by SDS-PAGE using 4–20% SurePAGE, Bis-Tris precast protein gels (GenScript, China) and transferred to Immun-Blot^® PVDF membranes (Millipore, United States). Membranes were blocked with 5% non-fat milk or 5% BSA in 1x TBS buffer containing 0.05% Tween 20 (TBST), incubated with primary and secondary antibodies diluted according to manufacturers’ instructions, and washed five times with ×1 TBST. Protein bands were visualized with Immobilon Western Chemilum HRP Substrate (Millipore, United States) on a BioSpectrum imaging system (Ultra-Violet Products, China).

hVSMCs were infected with either RAd60 or RAd-T3 at 600 pfu cell^-1 for 48 h, washed in cold PBS and lysed by scraping into 250 μl Immuno-Precipitation (IP) buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM EDTA, 10% v/v glycerol, 1% v/v NP40, 1% v/v Triton-X100, 2 x Complete^TM proteinase inhibitor cocktail (Roche GmbH) and phosphatase inhibitor cocktail II (Invitrogen). Lysates were centrifuged at 6000 x g for 5 min to remove remaining debris and split into 50 μl aliquots which were diluted to 500 μl with ice cold IP buffer, and pre-cleared with 50 μl of a 50% v/v protein-G sepharose/IP buffer slurry (Sigma Aldrich, UK) for 1 h. After centrifugation at 300 x g for 5 min to remove the protein-G, immunoprecipitation (IP) was performed by addition of PBS, 2 μg FAS antibody (3D5, Alexis/Enzo Scientific) or mouse IgG control antibody (Jackson ImmunoResearch, Stratech, UK) and incubated with gentle agitation at 4°C for 2 h. 60 μl 50% v/v protein-G/IP buffer slurry was then added for 30 min at 4°C. The protein-G immuno-complexes were centrifuged at 300 x g for 5 min, washed three times with 500 μl ice cold IP buffer before boiling in Laemlli buffer and resolving on 10% SDS-PAGE. After transfer to nitrocellulose, blots were probed for caspase-8 (S-19) and FAS (C-20) and visualised by ECL.

Proteins were isolated using lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol) supplemented with 1X cOmplete Proteinase Inhibitor Cocktail and 1X PhosSTOP Phosphatase Inhibitor Cocktail (both from Roche Diagnostics, Mannheim, Germany). Lysates were incubated on ice for 30 min and afterwards centrifuged at 21,250× g for 30 min at 4 °C. Western blot was performed as described elsewhere [47 (link)]. Antibodies used were anti-CD90/THY1 antibody (ab92574, Abcam, Cambridge, UK); anti-UCP1 (MAB6158, R&D, Minneapolis, MN, USA); hFAB rhodamine anti-actin and anti-tubulin (#12004164 and #12004166, BioRad).

Plants coexpressing hemagglutinin (HA)-labeled CDF2 (pCDF2::CDF2-HA) and yellow fluorescent protein (YFP)-labeled DCL1 (pDCL1::DCL1-YFP) were generated by crossing pDCL1::DCL1-YFP/dcl1-9 [26 (link)] with pCDF2::CDF2-HA/cdf2 which was obtained by crossing pCDF2::CDF2-HA/Col with cdf2 mutant. For a control, plants co-expressing pCDF2::CDF2-HA/cdf2 and p35S::YFP were generated by transforming the pCDF2::CDF2-HA/cdf2 line with the p35::YFP construct. The homozygous lines were grown in soil for 22 days, then seedlings were ground in liquid nitrogen and homogenized in three volumes of extraction buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 0.5% TritonX-100, 0.2% 2-mercaptoethanol, 5% glycerol) containing complete proteinase inhibitor cocktail (Roche) using a mortar and pestle set [50 ]. Cell debris was pellet by centrifugation for 10 min at 15,000 rpm. The Co-IP experiments were performed using HA Tag IP/Co-IP Kit according to the manufacturer’s protocol (Thermo Pierce). Briefly, the mixed lysate was incubated with anti-HA agarose for 4 h to overnight in each spin column. The columns were washed five times with TBS plus 0.05% Tween-20 detergent (TBS-T), and resolved by SDS/PAGE. Anti-GFP (Sigma) and anti-HA (Cell signaling technology) antibodies were used to detect DCL1-YFP and CDF2-HA, respectively.

Cells transfected with vector control and full-length ADAM15 were washed with ice-cold PBS, scraped off the tissue culture flask and lysed in 10 mM HEPES, pH 7.0, 150 mM NaCl, 5mM EDTA, 1% Triton X-100 and complete proteinase inhibitor cocktail (Roche Diagnostics; 10 μl/ml lysis buffer) as described above for 1 hour at 4°C. Protein concentration was determined and diluted to a concentration of 4mg/ml. For co-immunoprecipitation experiments, the mouse monoclonal anti-ADAM15, the goat polyclonal anti-ADAM15, that co-immunoprecipitated identical bands, were incubated with the freshly prepared cell lysates and Protein G conjugated agarose beads (Pierce) under constant agitation for 90 minutes at 4°C. The beads were washed 4 times with lysis buffer, boiled in sample buffer (125 mM Tris/HCl, pH 6.8, 4% SDS, 40% Glycerol, 10% β-mercaptoethanol, 20 μg/ml bromophenol blue) for 5 minutes at 95°C and subjected to SDS/PAGE. Subsequent Western Blotting was performed as described above.

ADAM15 transfected cells and HeLa cells were grown to subconfluency in petri dishes, and after washing twice with PBS incubated with the membrane impermeable EZ-Link Sulfo-NHS-LC-LC-Biotin from Pierce (0.1 mg/ml in PBS, pH 8.0, 0.9 mM CaCl₂, 0.5 mM MgCl₂) for 15 min at room temperature. After washing twice with PBS, cells were fixed with 1% PFA in PBS for 5 min at room temperature, and then quenched with 2.5 M glycin for 5 min. Cells were scraped off and harvested by centrifugation at 3000 xg for 5 min at 4°C and the cell pellet washed twice with PBS and lysed in 10 mM HEPES, pH 7.0, 150 mM NaCl, 5mM EDTA containing 1% Triton X-100 and complete proteinase inhibitor cocktail (Roche Diagnostics) and ultrasonicated on ice. Cell debris was removed by centrifugation at 13.000 rpm for 5 min, and the supernatant incubated with streptavidin conjugated magnetic beads (10 μl beads / 500 μg cell lysate; Thermo Fisher) and 1U benzonase nuclease (Merck) under rotation for 2h at room temperature. The magnetic beads were separated using a magnet, washed thrice with PBS/0.1% Triton X-100, and boiled in 20 mM Tris/HCl, pH 6.8, 0.5% SDS, 10% Glycerin v/v, 0,1% bromophenol blue w/v, 1% β-Mercaptoethanol for 15 min at 99°C to revert the PFA crosslinked protein complexes.