HAP1 parental fibroblasts were seeded into 24-well culture plates (CytoOne CC7682–7524) and grown to 75% confluence. Cells were then trypsinized, followed by recovery in growth medium on an orbital shaker for 10 min. Cells were pelleted at 200xg, washed once in PBS, and then lysed using Cell Lytic M reagent (Sigma C2978) containing 1X protease inhibitor cocktail (Sigma P8340) for 45 min on ice. Cell lysates were centrifuged for 10 min at 15000 rpm at 4C. The supernatant (soluble fraction) was diluted in LDS Sample buffer (Life Technologies #NP0007) and Reducing buffer (Life Technologies #NP00009). The pellet (insoluble fraction) was the resuspended in LDS Sample buffer and Reducing buffer. The two fractions were boiled at 95C for 5 min and subsequently analyzed by western blotting. Note that the actual fractions obtained by this method may vary from previous reports (Schiffhauer et al., 2019 (link)) since we did not use ATP in our lysis buffer, which would promote release of MII from the insoluble fraction into the soluble fraction. Therefore, our values for the insoluble fraction tend to be higher.
Cell lytic m reagent
Manufactured by Merck Group
Sourced in United States
Cell Lytic M is a reagent used for the lysis of mammalian cells. It is designed to effectively disrupt cell membranes and release the cellular contents, including proteins, for subsequent analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
P0217, by Agilent Technologies (2 mentions)
P0447, by Agilent Technologies (2 mentions)
Bradford method, by Bio-Rad (2 mentions)
Reducing buffer, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Genetools program, by Syngene (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Nb100 2220, by Novus Biologicals (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
9 protocols using cell lytic m reagent
1
Subcellular Fractionation and Western Blotting
2
T-cell Protein Extraction and Quantification
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Splenocytes were first isolated to sort total CD3+ T cells. Whole T‐cell extracts were obtained through using the Cell Lytic M reagent (Sigma). Extracts were separated by electrophoresis, transferred onto Hybond‐ECL membrane, and probed using primary antibodies. Densitometry analysis was conducted by first imaging the chemiluminescent blots with the ImageQuant LAS 4000 imager (GE Healthcare Life Sciences, Mississauga, ON, Canada) followed by analysis using the ImageQuant TL software (GE Healthcare Life Sciences).
3
Western Blot Protein Quantification
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Cell pellets were lysed with Cell Lytic M reagent (Sigma, MI, USA)) containing a protease inhibitor cocktail (Sigma, MI, USA). Proteins in crude lysates were quantified using the bicinchoninic acid assay (BCA) Protein Assay (Pierce Biotechnology, MA, USA)). A total of 20 μg of whole-cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Merck Millipore, Madrid, Spain). Blots were probed using primary antibodies (Table S3 ). Proteins were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-mouse (P0447, Dako, Barcelona, Spain) or anti-rabbit (P0217, Dako, Barcelona, Spain), incubated for 1 h at room temperature. Band intensity on the blots was quantified using the GeneTools Program (SynGene, Cambridge, UK).
4
Protein Extraction and Western Blotting
5
Preparation of Whole Cell Extracts
6
Vinculin Knockdown in Cell Culture
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Smart Pool Accell siRNAs against vinculin (E-009288-00-0005) and scrambled control (D-001910-10-05) were purchased from GE Dharmacon. Knockdown experiments were performed in 24 well plates using Lipofectamine 2000 (1690146, Life Technologies) according to the instructions provided by the manufacturer. Knockdown was performed for 72 hours, following which cells were plated on the growth substrate. For western blotting, cells were washed with PBS and lysed using Cell Lytic M reagent (Sigma) containing 1X Protease Inhibitor Cocktail.
7
Quantification of LC3 Lipidation
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The LC3-I to LC3-II processing assay was performed by lysing cells plated in 6-well culture dishes using CellLytic M reagent (Sigma-Aldrich Co. LLC, C2978) as per the manufacturer’s instructions, spinning down at 13,000 × RCF for 20 min, followed by SDS-PAGE of the supernatant fraction. We blotted using antibodies against TUBB (β-tubulin) (Developmental Studies Hybridoma Bank, E7), GFP (JL-8) (Clontech, 632380), and LC3 (Novus Biologicals, NB100-2220).
8
Protein Extraction and Western Blotting
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Protein lysates were prepared using the CellLytic-M reagent (Sigma-Aldrich) according to the manufacturers directions. Proteins were quantified by the Bradford method (Bio-Rad) and Western blotting was carried out using 25–50 μg of protein resolved by SDS-PAGE, transferred to PVDF membrane and immunoblotted as indicated. β-actin was used to assess protein loading.
9
Western Blot Analysis of Cell Lysates
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Cell pellets and EVs were lysed in Cell Lytic M reagent (Sigma-Aldrich, St. Louis, MO) and protein quantified using the bicinchoninic acid assay (BCA; Pierce Biotechnology, Waltham, MA). Twenty micrograms of of whole-cell lysates were separated by SDS-PAGE and transferred onto PVDF membranes (Merck Millipore, Burlington, MA). Membranes were blocked in 5% nonfat dry milk (w/v) in TBS buffer, 1 hour at RT. Targeted proteins were probed using primary antibodies (Table S2), O.N. at 4°C. Horseradish peroxidase (HRP)conjugated secondary antibodies, anti-mouse (P0447, Dako) or anti-rabbit (P0217, Dako), were then added and incubated for 1 hour at room temperature. Membranes were developed using Immobilion HRP substrate (Merck Millipore) in a LI-COR Odyssey Fc imaging system. Band intensity on the blots was quantified using the ImageJ NIH software.
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