Computational protocol: Paclitaxel inhibits selenoprotein S expression and attenuates endoplasmic reticulum stress

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[…] To determine the expression of associated proteins, the whole-cell lysates were prepared and western blotting was performed. Briefly, cells were harvested, resuspended in cell lysis buffer (1% Triton X-100, 0.015 M NaCl, 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and 10 µg/ml pepstain A) and then incubated on ice for 30 min. The cell lysates were centrifuged at 12,000 x g for 10 min at 4°C, and the supernatants were mixed with one quarter volume of 4x sodium dodecyl sulfate (SDS) sample buffer, boiled for 10 min, and then separated by 12% SDS-polyacrylamide gel electrophoresis. Following electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology) and blocked with 5% non-fat dry milk in Tris-buffered saline-Tween (TBST) buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl and 0.05% Tween-20) for 2 h at room temperature. Subsequently, the membranes were probed with the following primary antibodies in TBST buffer overnight at 4°C: Mouse monoclonal anti-SelS (1:1,000; sc-365498; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); rabbit polyclonal anti-GRP78 (1:1,000; sc-13968; Santa Cruz Biotechnology, Inc.); mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000; KC-5G4; Kangcheng Biotech, Shanghai, China). The membranes were then washed three times with TBST, and incubated with horseradish peroxidase-conjugated goat anti-mouse (1:2,000; A0216; Beyotime Institute of Biotechnology) and goat anti-rabbit (1:2,000; A0208; Beyotime Institute of Biotechnology) secondary antibodies for 30 min at room temperature, and washed extensively with TBST. Finally, protein bands were identified using enhanced chemiluminescence detection (ECL-Plus kit, Beyotime Institute of Biotechnology) and were digitally captured (MicroChemi, DNR Bio-imaging Systems, Ltd.). Differences in protein loading were controlled by blotting for GAPDH.Immunoreactive bands were quantified by densitometry of unsaturated images with background density subtracted (ImageJ; National Institutes of Health, Bethesda, MD, USA). GRP78 and SelS immunoblotting intensities were normalized by dividing by the corresponding GAPDH immunoblotting intensities from the same sample labeled on the same gel. Each experiment was repeated a minmum of three times to obtain the mean values and standard deviations. The normalized intensity values were statistically analyzed with SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA) using one-way analysis of variance followed by Dunnett's post-hoc tests. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Glucose, Picrotoxin, Tunicamycin, Paclitaxel