M per mammalian protein extraction reagent kit

The experiment was carried out according to the method described by Kim et al. [16 (link)] with modifications. RAW 264.7 cells were seeded in 10 cm cell-culture dishes at a density of 3 × 10⁶ cells per dish and cultured overnight. Cells were then pretreated with CuEO (0.001%, 0.01%) for 1 hour and stimulated with LPS (1 ug/mL) for 30 min. After incubation, the cells were collected and washed twice with cold PBS. Total cellular proteins were extracted by M-PER Mammalian Protein Extraction Reagent kit (Thermo Scientific). Cytoplasmic and nuclear proteins were extracted by using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific). Proteins were then maintained at −80°C for western blot analysis. Protein concentrations in the supernatant fractions were determined using a BCA protein assay (Sigma); then, proteins were applied to sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene fluoride membranes. After blocking for 60 min in a 5% skim milk solution, membranes were incubated overnight at 4°C with primary antibodies against P-JNK, JNK, p-ERK, ERK, p-p38, p38, and NF-κB p65, followed by horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The reactive bands were visualized by enhanced chemiluminescence.

Cells were disrupted using a M-PER Mammalian Protein Extraction Reagent kit (Thermo SCIENTIFIC) and total proteins were quantified using Bradford Assay. Total proteins (50ug) were used for western-blot analysis, and were loaded onto 10–12% SDS-PAGE (Mini-protean TGX Stain-free gels, BioRad, Hercules, California, USA) with prestained protein standards. Primary antibodies (mTOR, PTP1B, PTEN, TCTP, LC3B, p62, Caspase 3 and β-actin) were purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-core antibody from Enzo Life Science (Postfach, Lausen, Switzerland). Proteins were detected by chemiluminescence, according to manufacturer’s instructions (WesternBright^™ ECL, Advansta, Menlo Park, California, USA). Image analysis and quantification was performed using ChemiDoc^™ MP Imaging System and ChemiDoc^™ XRS+ software (BioRad).