Samples were prepared from the cells lysed with CelLyticTM M cell lysis reagent (Sigma-Aldrich, St. Louis, MO) supplemented with a protease inhibitor (cOmpleteTM protease inhibitor cocktail, Roche Applied Science). Whole cell lysates, fractionated cell lysates or immunoprecipitated samples were separated by SDS-PAGE and blotted to nitrocellulose membrane. Nitrocellulose membrane was firstly incubated with each primary antibody as described in the above Antibodies section, and then protein bands were detected by incubating secondary antibody: horseradish peroxidase (HRP)-conjugated antibodies (GE Healthcare, Little Chalfont, UK) and visualizing with enhanced chemiluminescence (Thermo Fisher Scientific) and Prime enhanced chemiluminescence (GE Healthcare, GE Healthcare, Little Chalfont, UK). Nuclear and cytoplasmic fractions were prepared from SNU449 and SNU475 cells 72 h after siRNA transfection and were prepared from 293T cells 48 h after transfection of an indicated expression vector(s). All the samples were fractionated by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacture's protocol.
Cellytic m cell lysis reagent
Manufactured by Merck Group
Sourced in United States, United Kingdom, Macao, Sao Tome and Principe, Japan
CelLytic M Cell Lysis Reagent is a proprietary detergent-based buffer solution designed for the gentle lysis of mammalian cells. It is formulated to extract soluble proteins from cell cultures or tissue samples while preserving the native structure and function of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (11 mentions)
Protease inhibitor cocktail, by Merck Group (9 mentions)
Laemmli sample buffer, by Bio-Rad (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Odyssey infrared imaging system, by LI COR (6 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Lane marker reducing sample buffer, by Thermo Fisher Scientific (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Anti flag m2 affinity gel, by Merck Group (4 mentions)
Enhanced chemiluminescence system, by PerkinElmer (4 mentions)
M iggκ bp hrp, by Santa Cruz Biotechnology (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Ecl plus, by GE Healthcare (3 mentions)
Horseradish peroxidase hrp conjugated antibody, by GE Healthcare (3 mentions)
Chemidoc mp system, by Bio-Rad (3 mentions)
Nupage gel, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
136 protocols using cellytic m cell lysis reagent
1
Western Blot Analysis of Cellular Fractions
2
Immunoblotting and Fractionation Protocol
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Cells or tissue samples were lysed using CelLyticTM M Cell Lysis Reagent (Sigma, C2978) and immunoblotting was performed as per manufacturer’s guidelines (Bio-Rad Laboratories, United States). Nuclear fractionation was done using NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833). Image acquisition was done using ChemiDoc (Bio-Rad ChemiDocTM MP System, 1708280). Densitometry analysis was performed using ImageJ software (NIH, Bethesda, MD, United States). Antibodies used were anti-ULK1 (Cell Signaling Technology, #8054), anti-GAPDH (Cell Signaling Technology, #5174), anti-phospho AKT (Cell Signaling Technology, #4060), anti-AKT (Cell Signaling Technology, #4685), anti-FoxO3a Ser253 (Cell Signaling Technology, #12829), anti-phospho FoxO3a (Cell Signaling Technology, #9466), anti-Beclin (Cell Signaling Technology, #3495), anti-ATG5 (Cell Signaling Technology, #12994), anti-SREBP2 (ABCAM, #ab30682), anti-HMGCR (ABCAM, #ab174830).
3
Western Blot Analysis of Apoptosis Regulators
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Proteins were extracted with CelLyticTM M Cell Lysis Reagent (Sigma‐Aldrich, Dorset, UK) supplied with protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, Dorset, UK). Proteins were mixed with NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min before analysis by western blot analysis. Proteins were subjected to 4–20% NuPAGE gels (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Sigma‐Aldrich, Dorset, UK) at 20 V for 1 hr by semidry transfer. PVDF membrane was blocked with 5% nonfat milk in TBST for 1 hr and then incubated with primary antibodies overnight at 4°C against the following targets: Bcl‐2 (100), RelA (NF‐κB p65, F‐6), Bcl‐XL (H‐5), Bax (2D2), Survivin (D‐8), and LDH (H‐10) (Santa Cruz Biotechnology, Inc., Dallas, TX); β‐actin (AC‐74) (Sigma‐Aldrich, Dorset, UK); p‐RelA (phospho‐NF‐κB p65‐Ser536), p‐STAT3 (phosphor‐STAT3‐Tyr705), p‐AKT, AKT, Mcl‐1, and Rb antibodies (Cell Signaling Technology‐New England Biolabs, Hitchin, UK). Bound antibodies were detected using appropriate horseshoeradish peroxidase‐conjugated secondary antibodies and visualized by GeneSnap (SynGene, Cambridge, UK) after adding ECL plus (GE Healthcare Life Science, Hatfield, UK).
4
Quantification of EMT and Stemness Markers
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Total protein of LPS tissue and cells were lysed by T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Cat. No 78510) or CelLytic TM M cell lysis reagent (Sigma-Aldrich, Cat. No C2978) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Cat. No 78438 and 78428), separately. Equal amounts of proteins were subjected to SDS-PAGE gel and transferred onto nitrocellulose membrane. After blocking with 5% BSA, the membranes were incubated with primary antibodies specific for AKT1 (2938S), AKT2 (3063S), AKT3 (14982S), p-AKT (T308) (13038S), p-AKT (S473) (4060S), pan-AKT (4685S), IWS1 (5681S), E-cadherin (3195S), Occludin (91131S), N-cadherin (13116S), Vimentin (5741S), Slug (9585S), Snail (3879S), KLF4 (12173S), Sox2 (2748S), OCT4 (2750S), Nanog (3580S), and β-actin (8457S) were purchased from Cell signaling and then incubated with IRDye 800CW-conjugated secondary antibodies (1:5000, LI-COR Biosciences, Cat. No 926-32211). Protein bands were visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences). Antibody specific for p-IWS1 was kindly gifted by Dr. Philip Tsichlis [27 (link)].
5
Immunoprecipitation of FLAG-tagged Proteins
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For whole-cell extract, transfected 293T cells were lysed by CelLyticTM M cell lysis reagent (Sigma-Aldrich, St. Louis, MO) and a subsequent immunoprecipitation reaction was conducted. Before lysing the cells, a protease inhibitor (cOmpleteTM protease inhibitor cocktail, Roche Applied Science) was added to cell lysis reagent under instructions of the manufacture. For fractionated cytoplasmic and nuclear components, immunoprecipitation reaction was conducted directly after cell fractionation. In an immunoprecipitation reaction, 300 μg of cell extract were incubated with 30 μL of anti-FLAG M2 affinity gel (Sigma-Aldrich, #A2220), which is a purified monoclonal anti-FLAG antibody covalently attached to agarose beads. After the beads were washed 3 times in 1 ml of TBS buffer (pH 7.6), proteins that bound to the beads were eluted by boiling in Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific).
6
Western Blot Analysis of Aquaporin-1 Expression
7
Western Blot Protein Analysis
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Proteins were extracted using CelLyticTM M cell Lysis Reagent (Sigma) supplied with protease and phosphatase inhibitor cocktails (Sigma). Proteins were subjected to 4–12% NuPAGE gels (Thermo Fisher Scientific) and transferred onto PVDF membrane (Sigma) at 20 V for 1 h by a semi-dry transfer. PVDF membrane was blocked with the blocking buffer [5% polyvinyl pyrrolidone PVP, 5% foetal calf serum and 0.1% sodium azide in tris-buffered solution (TBS) containing 0.2% Tween-20] for 30 min and then incubated with primary antibodies (Supplementary Table 2 ) overnight at 4 °C. Bound antibodies were detected using incubation with horseradish peroxidase-conjugated secondary antibodies in TBST, described previously50 (link).
8
Protein Extraction and Western Blot Analysis
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Proteins were extracted with CelLyticTM M cell Lysis Reagent (Sigma) supplied with protease inhibitor and phosphatase inhibitor cocktails (Sigma). Protein concentration was determined by the Bradford method, using the Bio-Rad Bradford reagent. Proteins were mixed with NuPAGE LDS Sample Buffer (Invitrogen) and boiled for 5 min. Proteins were then subjected to 4-20% NuPAGE gels (Invitrogen) and transferred onto PVDF membrane (Sigma) at 20 V for 1 hour by semi-dry transfer. PVDF membrane was blocked with 5% non-fat milk in TBST for 1 hour and then incubated with primary antibodies overnight at 4°C against the indicated targets. Bound antibodies were detected using appropriate HRP-conjugated secondary antibodies (Santa Cruz), visualized by GeneSnap (SynGene, Cambridge, UK) after adding ECL plus (GE Healthcare Life Science) [60 (link), 61 (link)].
9
Protein Turnover Analysis in Y1 Cells
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The expression plasmid of 3xFLAG-SF-1 (2.5 μg) was transfected into Y1 cells. 16 h after transfection in the presence or absence of CQ (100 μM), cells were treated with 50 μg/ml cycloheximide for different time periods and then harvested by CelLyticTM M cell lysis reagent (Sigma, St, Louis, MO) with cocktail protease inhibitors. Cell extract was cleared by centrifugation for 10 min at 13000 rpm and supernatant was analyzed by immunoblotting.
10
Cell Lysis and Immunoprecipitation
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Transfected 293T cells were lysed with CelLyticTM M cell lysis reagent (Sigma-Aldrich) containing a complete protease inhibitor cocktail (Roche Applied Science). In a typical immunoprecipitation reaction, 300 μg of whole-cell extract was incubated with an optimum concentration of primary antibody. After the beads had been washed 3 times in 1 ml of TBS buffer (pH 7.6), proteins that bound to the beads were eluted by boiling in Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific).
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