Cells (5 × 105 to 10 ×105) were lysed with Cell Lysis Buffer (10X) (Cell Signaling Technologies, 9803) with protease inhibitors. The samples were loaded with 5× denaturing sample buffer and separated by 12% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride membranes and were subsequently analysed by immunoblot with the relevant antibodies. The blots were developed by using chemiluminescence (protein simple, Fluorchem E FE0605). The monoclonal antibody used to detect β-actin was from Santa Cruz (sc-47778), and the polyclonal antibody to TLR3 was from Novus Biologicals (NBP2-24565).
Cell lysis buffer 10x
Manufactured by Cell Signaling Technology
Sourced in United States
Cell Lysis Buffer 10X is a concentrated solution used to lyse cells and extract their cellular contents. It is a fundamental tool for protein extraction and subsequent analysis.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail tablet, by Roche (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail tablet, by Roche (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sc 47778, by Novus Biologicals (1 mentions)
Nbp2 24565, by Novus Biologicals (1 mentions)
Ab14933, by Abcam (1 mentions)
Ab76252, by Abcam (1 mentions)
Ab94744, by Abcam (1 mentions)
Ab52771, by Abcam (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Pmsf 93, by Merck Group (1 mentions)
Gapdh d16 h11 xp 5174, by Cell Signaling Technology (1 mentions)
Anti osteopontin antibody ab8448, by Abcam (1 mentions)
Raf 1, by Cell Signaling Technology (1 mentions)
B raf, by Cell Signaling Technology (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
Chemidoc xrs imager, by Bio-Rad (1 mentions)
16 protocols using cell lysis buffer 10x
1
Immunoblot Analysis of TLR3 Expression
2
Protein Expression Analysis in Osteogenic Cells
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Cell cultures were lysed with Cell lysis buffer (Cell Lysis Buffer (10X) #9803 Cell Signalling Technology) containing 10 mM phenylmethylsulphonyl fluoride (PMSF 93,482 Sigma) as a protease inhibitor. Lysates were incubated on ice for 15 min and centrifugated at 13 000 × g for 20 min at 4°C. Equal micrograms (20 µg) of proteins quantified with bicinchoninic acid (BCA) assay (Thermo Scientific) and boiled in SDS sample buffer (2x Laemmli Sample Buffer BIORAD cat.#161-0737) were resolved on 10% SDS-PAGE and transferred to PVDF membranes (Immun-Blot® PVDF Membrane for protein Blotting BIORAD cat.#162-0177). Blots were blocked for 1 h in PBS-T (PBS plus 0.05% Tween-20), 5% non-fat, dried milk and probed overnight at 4°C with DEPTOR/DEPDC6 antibody, NBP1-49,674 (Novusbio); GAPDH (D16 H11) XP #5174 (Cell Signalling Technology); Anti-YAP (phosphor S127) antibody [EP1675Y] ab76252 (Abcam); anti-YAP antibody [EP1674Y] ab52771 (Abcam); Anti-Sp7/Osterix antibody ab94744 (abcam); anti-BMP2 antibody ab14933 (abcam); RUNX2 (D1L7 F) Rabbit mAb #12,556 (Cell Signalling Technology); Anti-Osteopontin antibody ab8448 (abcam). Immunocomplexes were detected with horseradish peroxidase-conjugated species-specific secondary antibodies (Santa Cruz Biotechnology) followed by enhanced chemiluminescence reaction with Immobilon Western Chemiluminescence HRP substrate WBKLS0100 (Millipore).
3
Protein Analysis of MAPK Signaling Pathway
4
Immunoprecipitation and Kinase Activity Assay
5
Biochemical Reagents for Cell Culture
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Gallic acid, quercetin, dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12 medium, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Geneticin (G418) was purchased from Invitrogen (San Diego, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Bio Basic (Markham, Ontario, Canada). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Phosphate buffer saline (PBS) was HyCloneTM and purchased from GE Healthcare Bio-Sciences, USA. EmbryoMax® non-essential amino acids (NEAA) solution was purchased from Millipore® (Burlington, MA, USA). Penicillin/Streptomycin solution was purchased from Gibco (Waltham, MA, USA). Cell lysis buffer (10X), anti-APP, anti-β-actin, and horseradish peroxidase-coupled secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Methanol was purchased from Merck (Darmstadt, Germany).
6
Quantitative Fibronectin Protein Analysis
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The proteins were isolated from the snap-frozen tissues using Cell Lysis Buffer (10X) (Cell Signaling Technology, USA) supplemented with 1 mM PMSF (Cell Signaling Technology, USA) and protein concentration was determined using the Bradford assay. Equal amounts of proteins were boiled in SDS Sample Loading Buffer, separated on 7.5% SDS-PAGE gel, and transferred to a 0.2 μm nitrocellulose membrane (Amersham, UK). After blocking the membrane with 1X ROTI (Carl Roth, Germany) for one hour, the membrane was washed with Tris-buffered saline and Tween 20 (TBS-T) and then incubated at 4 °C overnight with anti-fibronectin antibody (F3648, Sigma-Aldrich, USA). Anti-β-tubulin antibody (ab6046, Abcam, USA) was used as housekeeping. The next day, the membrane was washed with TBS-T and then incubated with a secondary Goat anti-Rabbit IgG Alexa Fluor™ 680 antibody (A21109, Invitrogen, USA). Proteins were visualized by an Odyssey Imaging System (LI-COR Biosciences, USA) and relative intensities of bands were evaluated using the Image Studio software (LI-COR Biosciences, USA).
7
Cell Culture and Molecular Reagent Acquisition
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Cell culture media Dulbecco's Modified Eagle's medium (DMEM), fetal calf serum (FCS), phosphate buffer saline (PBS), penicillin and streptomycin were purchased from PAA Laboratories, Pasching, Austria. Fluorescein isothiocyanate-conjugated dextran (FITC-dextran), 4 kDa (FD4), Trypsin, MPA, and DMSO were purchased from Sigma-Aldrich, Steiheim, Germany. Protease and phosphatase inhibitor cocktails were purchased from Roche, Mannheim, Germany. Bromophenol blue was obtained from Carl Roth, Karlsruhe, Germany. Sodium dodecyl sulfate (SDS) was obtained from Serva, Heidelberg, Germany. Glycerin, potassium ferricynaide, and sodium thiosulfate were purchased from Merck, Darmstadt, Germany. Formic acid was purchased from BASF, Ludwigshafen, Germany. Magnesium chloride (MgCl2), M-MLV RT enzyme, and 5X PCR buffer were from Invitrogen, Karlsruhe, Germany. Deoxynucleotide triphosphates (dNTPs) were from Roche, Mannheim, Germany and PCR primers were synthesized by Eurofins, Ebersberg, Germany. Ribonuclease (RNAase) inhibitor was obtained from Promega, Mannheim, Germany. The cell lysis buffer (10X) was obtained from Cell Signaling Technology, Danvers, MA, USA. All other chemicals used in this work were from the highest available purity from commercial sources unless otherwise stated.
8
P. aeruginosa Infection of Lung Cells
9
Investigating Combination Treatments for Cancer Cell Cytotoxicity
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HEK293 cells (Vaccera, Egypt) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax-1, sodium pyruvate, and 1 mg/ml glucose supplemented with 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mm glutamine, and 10% (v/v) fetal calf serum. Cells (1x106) were seeded in T-75 cm2 flask and incubated at 37°C in 90% air/10% CO2 for 3 days to reach 70% confluence [24 (link)]. Cells were treated with one of the following treatments for 24 hr (5 replicates): (i) DMSO, (ii) cisplatin 14 μM [24 (link)], (iii) cisplatin + pioglitazone 10 μM [25 (link)], (vi) cisplatin + fenofibrate 20 μM [26 (link)], (v) cisplatin + fenofibrate + pioglitazone, (vi) cisplatin + thalidomide 100 μM [27 ], (vii) cisplatin + fenofibrate + pioglitazone + thalidomide. Cells were washed with phosphate buffered saline (PBS) and trypsinized. Cell pellets were re-suspended in 1 ml PBS and cell lysates were obtained using Cell Lysis Buffer 10X (Cell Signaling Technology Cat No. 9803) as described by the manufacturer [28 (link)], divided into 4 aliquots and stored at -70°C till used for biochemical determinations (TNF-α, IL-6, caspase-3, and SOD).
10
Protein Expression Analysis Protocol
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Protein crude was recovered by Cell Lysis Buffer (10X; #9803, Cell Signaling, Danvers, Massachusetts, USA), and protease inhibitors (Complete Mini, Roche). Lysates were cleared by centrifugation at 10,000 rpm for 10 min at 4 °C. The total protein concentration of the supernatant was determined using a BSA protein (Bio-Rad Laboratories, Des Plaines, IL, USA). Cell lysates (2–50 μg proteins) were applied on 12% acrylamide gel, transferred to PVDF membrane (Millipore, Immobilon), and then probed with one of the following primary antibodies (all mouse IgG, 1 µg/mL) overnight at 4 °C: C-terminal cytoplasmic domain of HB-EGF of mouse origin (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-1414), MMP7 (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-515702), β-actin (Cell Signaling, #4967), total p44/42 MAPK (ERK1/2)(Cell Signaling, #9102), p-EGFR (Santa Cruz Biotech, sc-57545), MMP9 (Chemicom, AB19047)), ERK1/2 (Cell signaling, 4370S), BAX2 (Cell signaling, Danvers, MA, USA, #2772S). Membranes were stained with secondary antibody conjugated with horseradish peroxidase (Nichirei, rabbit-HRP, or goat-HRP), and developed with the ECL Plus detection system (Amersham Life Science, RPN2132) using image analyzer Image-Quant LAS4000 (GE Healthcare, Uppsala, Sweden).
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