RNA was isolated from cellular samples using the RNeasy Mini Kit (Qiagen) as described by the manufacturer. Cells were collected by centrifugation, washed twice in ice-cold PBS, and resuspended in Buffer RLT containing 40 µM DTT. The resulting mRNA yield was determined on a Nanodrop Spect...
Cells were lysed in lysis buffer (25 mM HEPES, pH 7.6, 150 mM NaCl, 1% NP-40, 2% SDS and 10% sucrose) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphatase inhibitor cocktail (5 mM NaF, 200 μM sodium orthovanadate, 1 mM ...
Cells were washed with PBS, lysed in 1x SDS sample buffer, and boiled. The cell lysates were then centrifuged (13,200 rpm). The supernatants were collected and separated by electrophoresis on a 10% SDS-polyacrylamide gel, after which the separated proteins were transferred to a PVDF membrane...
Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton-100, 1 mM PMSF and protease inhibitor cocktail set I). Protein content was quantitated using Bradford assay. Equivalent amounts of total protein were resolved by 15% SDS-PAGE and transferred to ...
Following PBS/glycine washes, membranes were excised from transwells, and cells were lysed by shaking for 1 h in 1.5 ml of 2× PBS, 1% Triton X-100, 40 mm octyl glucoside. Lysates were diluted to 10 ml with 2× PBS, 0.5% Triton X-100, and cell debris was removed by centrifugation...
Cell pellets were lysed in appropriate volumes of lysis buffer to generate extracts containing equal number of cells per unit volume. A fluorometric based assay for caspase-3 activity was performed using Ac-DEVD-AMC as a substrate as described in the methods section. Bars are representative of ...
In brief, 1 × 106 cells were treated with metformin for 48 h and then lysed with lysis buffer to obtain supernatant as proteins. For each group, 50 µg of protein was mixed with 50 µl of 2X Reaction buffer (containing 10 mm DDT) and 5 µl DEVD-pNA. The mixtures were ...
The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in li
The cells were lysed in a 1% SDS solution. The absorbance of the solution was measured using a microplate spectrophotometer at a wavelength of 595 nm. Epigenome-wide DNA methylation Methylation profiles were obtained using the Illumina Infinium HumanMethylationEPIC BeadChip kit, which assayed ...
Cells were lysed directly in the transwell insert or the 6-well plate using SDS sample lysis buffer (4% SDS, 125 mM Tris–HCl pH 6.8, 20%, glycerol in double-distilled water) and sonicated using a Sonopuls GM 2070 tip sonicator (Bandelin) to fragment DNA and decrease viscosity. Protein ...