Protease inhibitor cocktail

Cells were lysed in Triton X-100 containing buffers (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% Triton X-100, Roche protease inhibitor cocktail, or the luciferase assay buffer stated below), in an SDS containing buffer (100 mM NaCl, 5 mM MgCl₂, 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 1% Triton X-100, 0.5% SDS, 10% glycerin, protease inhibitor cocktail) for better solubilization of Gα proteins, or in hypotonic lysis buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, Roche protease inhibitor cocktail) when assessing β-catenin levels. Protein samples were separated via polyacrylamide gel electrophoresis under denaturing or native conditions (no SDS), when indicated, and transferred onto a nitrocellulose membrane, which was probed with indicated primary and respective horseradish peroxidase (HRP)-conjugated secondary antibodies (see below). Protein bands were detected via light emission upon HRP-catalyzed oxidation of luminol in a LAS-3000 with Image Reader software (FUJIFILM) or a PXi6 (Syngene) with GeneSys software, and band intensities were quantified with AIDA 2D densitometry or Fiji-ImageJ when required.

The brains of 6- to 8-week-old rats were removed quickly and separated into four regions, including the cortex, hippocampus, striatum and cerebellum. Brain tissues were homogenized with a motorized tissue grinder (G50, Coyote Bioscience, Campbell, CA, USA) at 3,000 rpm in buffer 1 [10 mM HEPES (pH 7.4), 2 mM EDTA, 5 mM sodium orthovanadate, 30 mM sodium fluoride, 20 mM β-glycerolphosphate, and protease inhibitor cocktail (Roche)]. The total homogenates were centrifuged at 500× g for 5 min at 4°C to remove nuclei, extracellular matrix and cell debris. The supernatant was collected and centrifuged at 10,000× g for 15 min at 4°C to separate the crude membrane fraction pellet 2 (P2). P2 was resuspended in 300–500 μl buffer 2 [50 mM HEPES (pH 7.4), 2 mM EDTA, 2 mM EGTA, 5 mM sodium orthovanadate, 30 mM sodium fluoride, 20 mM β-glycerolphosphate, 1% Triton-X-100, and protease inhibitor cocktail (Roche)] and centrifuged at 20,000× g for 80 min at 4°C to obtain pellet 3 (P3). P3 (Triton X-100 insoluble PSD fraction) was resuspended in 50 μl buffer 3 [50 mM Tris (pH 9), 5 mM sodium orthovanadate, 30 mM sodium fluoride, 20 mM β-glycerolphosphate, 1% NaDOC, and protease inhibitor cocktail (Roche)] and frozen in liquid nitrogen for storage at −80°C.

3 × 10⁶ cells were trypsinized, washed twice in ice-cold PBS and lysed in five volumes of hypotonic lysis buffer (10 mM HEPES pH 7.9, 60 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 0.075% NP-40, 1× protease inhibitor cocktail (Roche)) for 10 min. Lysates were centrifuged at 500 × g for 5 min and resulting supernatant was collected as the cytoplasmic fraction. The nuclear pellet was washed gently for 3 times in 800 μl hypotonic lysis buffer without NP-40. Nuclei were lysed in one volume of nuclear lysis buffer (20 mM HEPES pH 7.9, 400 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 5% glycerol, 1× protease inhibitor cocktail (Roche)) and dilute with two volumes of dilution buffer (20 mM HEPES pH 7.9, 1.6% Triton-X-100, 0.2% sodium deoxycholate, 1× protease inhibitor cocktail (Roche)).

YenTc toxin particles from Y. entomophaga cultures were purified as previously published^{13 (link)}. Volumes of 50 ml of an overnight culture of Y. entomophaga WT and derivatives were pelleted. Supernatant was collected and filtered two times through a 0.2 μm filter. The supernatant was then subjected to ammonium sulfate precipitation to a final concentration of ~70% w/v ammonium sulfate. The resulting precipitate was resuspended in Tris buffer (25 mM Tris, 150 mM NaCl, protease inhibitor cocktail (Roche), pH 7.5). Another method of purifying YenTc particles was performed using lysis of the cell pellet. Here, cell pellets from the same cell culture were lysed for 1 h at 37 °C, shaking in the lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5x CellLytic B (Sigma-Aldrich), 1% Triton X-100, 200 µg ml⁻¹ lysozyme, 50 µg ml⁻¹ DNAse I, protease inhibitor cocktail (Roche), 5 mM MgCl₂, pH 7.5). Cell debris was removed by centrifugation (15,000g, 15 min, 4 °C) and cleared lysates were subjected to ultra-centrifugation (15,0000g, 1 h, 4 °C) with a 2 ml 40% sucrose cushion. Pellets were resuspended in 100 µl resuspension buffer (25 mM Tris, 150 mM NaCl, protease inhibitor cocktail (Roche), pH 7.5). Proteins in the toxin particle preparations were identified by mass spectrometry at the Functional Genomics Center Zürich (FGCZ).

Approximately 2.5 × 10⁶ cells were pelleted, washed with PBS, resuspended in 250 μl Lysis Buffer (15 mM HEPES pH7.5, 10 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM EGTA, 250 mM Sucrose, 0.4% Igepal, 1 mM DTT, 40 U/ml RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and incubated on ice for 20 min. Nuclei were centrifuged at 2,000 g for 10 min at 4°C and the supernatant was collected as the cytoplasmic fraction. Nuclei were then resuspended in 50 μl Nuclei Lysis Buffer (10 mM HEPES pH7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 40 U/ml RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and incubated on ice for 5 min. Nuclei were pelleted at 17,000 g for 5 min at 4°C and the supernatant was removed as the nucleoplasm fraction. The pellet was then resuspended in 50 μl Salt Extraction Buffer (25 mM HEPES pH7.5, 10% glycerol, 420 mM NaCL, 5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 40 U/ml RNaseOUT (Invitrogen), protease inhibitor cocktail [Roche]) and incubated for 30 min at 4°C with rotation. The sample was then centrifuged at 17,000 g for 20 min at 4°C. The supernatant was collected as the salt extracted fraction and the pellet resuspended in 50 μl Salt Extraction Buffer to generate the chromatin fraction. RNA was isolated from each fraction using the Qiagen Mini RNeasy kit following the manufacturer's instructions.

Protocol was adapted from the Bönisch lab (Arrigoni et al. 2016 (link)). Twenty million K562 cells were grown/experiment. Cells were crosslinked and pelleted as described for the whole-cell fRIP above. Crosslinked pellets were thawed and gently resuspended in 1 mL cold Farnham lab (FL) buffer (5mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1× Protease Inhibitor Cocktail [Roche]). Cells were then sonicated in a Covaris Ultrasonicator for 30 sec with 75W peak power, 2% duty factor and 200 cycles/burst at 4°C–10°C.
Cells were spun down at 1000g for 5 min at 4°C. Supernatant was removed and each nuclear pellet was washed once with 1 mL 1× FL buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1× Protease Inhibitor Cocktail [Roche]). Pellets were spun down at 1000g for 5 min at 4°C. Supernatant was removed and pellets were resuspended in 500 µL of 1× fRIP lysis buffer (50 mM Tris pH 8, 150 mM KCl, 0.1% SDS, 1% Triton-X, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5 mM DTT, 1× Protease Inhibitor Cocktail [Roche], 100 U/mL RNasin Plus RNase Inhibitor [Promega]). fRIP was then performed as described above for whole-cell pellets. Nuclear fractionation was confirmed by western blot. Antibodies used for western blot include: DNMT1 (Abcam 19905), EZH2 (Cell Signaling Technologies 5246S), and β-actin (Sigma-Aldrich A5441).