1–2 × 108 U2OS cells were grown in DMEM (Fisher Scientific 11995073) supplemented with 10% FBS, penicillin-streptomycin (Thermo Fisher 15140122) and GlutaMAX (Fisher Scientific 35050061) at 37 °C under 5% CO2. Cells were collected by trypsinization, pelleted at 500xg, and resuspended in 10 mL of buffer CE+NP40 (cytoplasmic extract) (20 mM HEPES, 10 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.1% NP40, 1 mM DTT, cOmplete protease inhibitor cocktail (Sigma 11873580001)). After 5 min incubation on ice, the sample was centrifuged at 500×g, 4°C. Pellet was washed three times in 4 mL of buffer CE (20 mM HEPES, 10 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, cOmplete protease inhibitor cocktail (Sigma 11873580001)) and resuspended in 1 mL of buffer NE (nuclear extract) (20 mM Tris-HCl, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF, 25% glycerol, 1 mM DTT, cOmplete protease inhibitor cocktail (Sigma 11873580001)). This sample was rotated at 4°C for 1 hour and clarified by centrifugation at 20,000×g. Clarified lysate was dialyzed in dialysis buffer (20 mM Tris-HCl, 75 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF, 10% glycerol, 1 mM DTT, cOmplete protease inhibitor cocktail (Sigma 11873580001)) using Slide-A-Lyzer™ MINI Dialysis Device (Fisher Scientific 88404). Extracts were clarified and protein concentration was measured by Qubit™ Protein Assay Kit (Thermo Fisher Q33211).
Complete protease inhibitor cocktail
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, United Kingdom, Macao, China, Japan, France, Canada, Italy
The Complete Protease Inhibitor Cocktail is a laboratory product designed to inhibit a wide range of proteases. It is a concentrated, ready-to-use solution that can be added directly to protein samples to prevent proteolytic degradation.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (40 mentions)
Phosstop, by Merck Group (29 mentions)
Ripa buffer, by Thermo Fisher Scientific (14 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Phosphatase inhibitor cocktail, by Merck Group (12 mentions)
Pvdf membrane, by Bio-Rad (9 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (9 mentions)
Ripa buffer, by Merck Group (9 mentions)
Complete protease inhibitor cocktail, by Roche (8 mentions)
Ripa lysis buffer, by Merck Group (8 mentions)
Odyssey infrared imaging system, by LI COR (8 mentions)
Nitrocellulose membrane, by Bio-Rad (8 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Image lab software, by Bio-Rad (7 mentions)
Mini protean tgx precast gel, by Bio-Rad (7 mentions)
β actin, by Merck Group (7 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (7 mentions)
Trans blot turbo transfer system, by Bio-Rad (7 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (7 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (6 mentions)
564 protocols using complete protease inhibitor cocktail
1
Isolation of Nuclear Extracts from U2OS Cells
2
Nuclei Extraction and Lysis Protocol
3
Nuclei Extraction and Lysis Protocol
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Nuclei were extracted by quick lysis in RSB-G40 lysis buffer (10mM Tris 7.4, 10mM NaCl, 3 mM MgCl2, 10% glycerol, 0.25% NP-40, 0.5 mM DTT, 25 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and cOmplete protease inhibitor cocktail (Sigma)). Nuclei were washed with RSB-G40 without NP-40, and lysed with high-salt extraction buffer (20 mM HEPES pH7.9, 420mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 25 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and cOmplete protease inhibitor cocktail (Sigma)). Salt concentrations were adjusted with the addition of binding buffer (20mM HEPES pH7.9, 5 mM MgCl2, 10% glycerol, 0.1% Tween-20, 0.5 mM DTT, 25 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and cOmplete protease inhibitor cocktail (Sigma)), and lysates were subjected to either electrophoresis or immunoprecipiation.
4
Immunoprecipitation of ATG9 in U2OS Cells
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Cell lysates were obtained in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, supplemented with 0.5 mM NaF, 2 mM EGTA, 1 mM sodium orthovanadate and complete protease inhibitor cocktail (Sigma). Lysates were clarified by centrifugation for 10 min at 4°C and protein concentrations were assessed using Bradford protein assay (BioRad Laboratories). Samples containing equal amounts of proteins were boiled in SDS sample buffer, resolved using SDS-PAGE and transferred to nitrocellulose membranes. The blots were then probed with the appropriate antibodies. Before immunoprecipitation, U2OS cells stably expressing HA-Flag-ATG9 were transiently silenced for CAPNS1 for 3 days, and then treated for 1 h with 100 nM thapsigargin. Whole cell lysates in 20 mM CHAPS, 125 mM NaCl, 50 mM Tris-HCl, pH 7.5, supplemented with 0.5 mM NaF, 1 mM Sodium orthovanadate, complete protease inhibitor cocktail (Sigma), were incubated for 2 h with anti-Flag antibody or with the anti-Myc antibody as negative control. Subsequently, protein G (GE Healthcare Life Sciences) was added for 2 h at 4°C. Samples were subjected to SDS-PAGE and immunoblotting.
5
Thylakoid Membrane Extraction: Optimized Protocols
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The thylakoid membrane extraction protocols further called “the conventional protocol” and “PVC protocol” (the PEG + vitamin C) were inspired by [38 (link)] with some modifications in the buffer composition. For optimization experiments using Posidonia oceanica, we used leaves from different growing depths. Detailed instructions about the complete extraction procedure are shown in Additional file 3 : Method S1. The composition of the buffers and the main step of the procedure are described below:
Thylakoid membranes were extracted by grinding leaves in two distinct buffers (GB). For the conventional protocol, the buffer was composed of 20 mM Tricine pH 7.8, 0.3 M sorbitol, 10 mM EDTA, 10 mM NaHCO3, 0.15% bovine serum albumin (BSA), 10 mM of phosphatase inhibitor NaF, 5 mM benzamidine, 5 mM caproic acid. For the PVC protocol, the grinding buffer described above was supplemented with 5% Polyethylene glycol 4000 (PEG4000) and 5% ascorbic acid (vitamin C, Sigma).
Chloroplasts were then burst using a hypotonic buffer (25 mM Hepes–KOH pH 7.5, 25 mM sorbitol, 5 mM NaCl, 5 mM MgCl2, 5 mM KCl, 10 mM NaF, and 5 mM Benzamidine, 5 mM caproic acid, cOmplete™ Protease Inhibitor Cocktail).
The extracted thylakoid membranes were stored in the storage buffer (50 mM Hepes pH 7.5, 0.3 M sorbitol, 10 mM NaCl, 5 mM MgCl2, 10 mM NaF, cOmplete™ Protease Inhibitor Cocktail).
6
Quantitative Western Blot Analysis of Protein Signaling
7
Isolation of Sarcoplasmic Membrane from Muscle
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The sarcoplasmic membrane was isolated in accordance with previously described methods (Parolini et al, 2009 ). Briefly, vastus medialis (VM) or tibialis anterior (TA) muscles were put in ice‐cold homogenization buffer (250 mM sucrose and 5 mM HEPES pH 7 in 0.2% NaN3) supplemented with complete protease inhibitor cocktail (Sigma‐Aldrich) and homogenized with an electric homogenizer. The homogenates were then centrifuged at 5,500 g for 10 min at 4°C. The supernatants were harvested and centrifuged at 12,500 g for 18 min at 4°C. The pellets were discarded, and the supernatants were centrifuged again at 12,500 g for 18 min at 4°C. The supernatants were then transferred into ultracentrifuge tubes and centrifuged at 50,000 g for 1 h at 4°C. The pellets were resuspended in homogenization buffer supplemented with 600 mM KCl and complete protease inhibitor cocktail (Sigma‐Aldrich) and incubated for 30 min on ice. The samples were then centrifuged at 15,000 g for 10 min at 4°C. The resulting supernatants were centrifuged at 50,000 g for 1 h at 4°C. The final pellets were dissolved in homogenization buffer. The protein content was determined by standard BCA assays as in Parolini et al (2009 ).
8
ChIP-seq Analysis of FLAG-KASH in Drosophila Brains
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Approximately 500 flies expressing FLAG-KASH in the MBs were used for ChIP-seq analysis, as previously described with some modifications43 (link). Heads were collected and homogenized in crosslinking buffer (15 mM Hepes-KOH at pH 7.5, 100 mM NaCl, 0.25 M sucrose, 0.1% NP40, 1 mM dithiothreitol, and 1 mM PMSF) containing 1% formaldehyde, and Complete Protease Inhibitor Cocktail (Sigma, St. Louis, MO, USA) using a Teflon/glass homogenizer. The homogenate was left on ice for 15 min. Crosslinking was quenched by adding 125 mM glycine, and the homogenate was filtered through 40 μm nylon mesh to remove cuticles. The nuclei were rinsed three times in ChIP buffer (20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) containing 0.25 M sucrose. The nuclei were then dissolved in ChIP buffer, containing 0.25 M sucrose, Complete Protease Inhibitor Cocktail and 1 mM PMSF, briefly sonicated to dissociate the individual nuclei, and immunoprecipitated with ANTI-FLAG M2 Affinity Gel (Sigma, St. Louis, MO, USA) for 2 h at 4 °C. The beads were rinsed four times in extraction buffer, with 5 min nutation at 4 °C between washes, and subjected to ChIP analysis.
9
Chromatin-Bound Protein Extraction
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Cells were lysed with buffer A (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES (pH 6.8), 1 mM EGTA, and 0.2% Triton X100, containing PhosSTOP (Merck, Darmstadt, Germany) and Complete Protease Inhibitor Cocktail (Merck, Darmstadt, Germany) for 8 min on ice. Lysates were centrifuged at 5000× g at 4 °C for 5 min to separate the chromatin-containing pellet. Supernatants were obtained as soluble fractions. The pellet was digested with 50 units of Benzonase (Enzynomics, Daejeon, Korea) for 40 min in RIPA buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, 1 mM PMSF, 5 mM MgCl2, containing PhosSTOP (Merck, Darmstadt, Germany) and Complete Protease Inhibitor Cocktail (Merck, Darmstadt, Germany)) to extract chromatin-bound proteins. The chromatin-containing fractions were clarified by centrifugation (13,000× g, 4 °C) for 5 min to remove debris. The protein concentration was determined using the Bradford Assay (Bio-Rad, Hercules, CA, USA), and the proteins were analyzed by immunoblotting.
10
Whole Cell, Cytoplasmic, and Nuclear Protein Extraction
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To obtain the whole cell extracts, cells were washed with ice-cold PBS and the cell pellet was reuspended in Tris lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol [DTT], 1 mM Na3VO4) with Complete protease inhibitor cocktail (Merck, USA).
For preparation of cytoplasmic and nuclear extracts, cells were harvested and resuspended in harvest buffer containing 10 mM HEPES (pH 7.9), 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, 0.5% Triton X-100 and with Complete protease inhibitor cocktail (Merck). After obtaining the cytoplasmic extract, the nuclear pellet was further washed with wash buffer/buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, and 0.1 mM EGTA) and then resuspended in buffer C (10 mM HEPES [pH 7.9], 500 mM NaCl, 0.1 mM EDTA, 0.1 mM egtazic acid [EGTA], 0.1% NP40) and protease inhibitor cocktail to extract the nuclear proteins.
For preparation of cytoplasmic and nuclear extracts, cells were harvested and resuspended in harvest buffer containing 10 mM HEPES (pH 7.9), 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, 0.5% Triton X-100 and with Complete protease inhibitor cocktail (Merck). After obtaining the cytoplasmic extract, the nuclear pellet was further washed with wash buffer/buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, and 0.1 mM EGTA) and then resuspended in buffer C (10 mM HEPES [pH 7.9], 500 mM NaCl, 0.1 mM EDTA, 0.1 mM egtazic acid [EGTA], 0.1% NP40) and protease inhibitor cocktail to extract the nuclear proteins.
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