Ecl western blot kit

Total cellular protein extracts were obtained with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% PMSF (Sigma) and 1% phosphatase inhibitor (Roche Applied Science). The concentration of protein was measured by the BCA protein assay kit (Thermo). Next, equal amounts of samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with skimmed milk in TBST buffer (0.1 M Tris, 150 mM NaCl, and 0.1% Tween-20), the membranes were incubated overnight at 4 °C with primary antibodies against HSPB7 (Abcam, Cambridge, UK), RUNX2 (Abcam), OCN (Abcam), ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), phosphorylated-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology), and GAPDH (HuaxingBio Science, Beijing, China). Subsequently, the membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. The bands were visualized via the ECL Western Blot Kit (CoWin Biotech). The intensity of bands was quantified using ImageJ analysis software (http://rsb.info.nih.gov/ij/) and normalized to GAPDH.

YOM38 yeast cells containing the pR316-GFP-ATG8 plasmid were cultured as described in the previous section. At first, yeast cells were incubated with 300 μM RES or CuB at doses of 0, 0.1, 0.3, or 1 μM, and the yeast cells were collected at the specified time point and washed three times with PBS. After that, the samples were ultrasonicated for five times (1 min for each time), freeze-thawed for five times, and sonicated for five times again. The cell lysates were centrifuged, and the protein concentrations of the supernatant were measured with BCA Protein Assay Kit (CoWin Biotech, Beijing, China). Approximately 20 μg protein was separated with SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies followed by secondary antibodies. Antigens were visualized using ECL Western Blot Kit (CoWin Biotech, Beijing, China). The primary antibodies used are as follows: anti-GFP antibody (Medical & Biological Laboratories, Nagoya, Japan) and anti-β-actin antibody (CoWin Biotech, Beijing, China). The secondary antibodies used are as follows: horseradish peroxidase-linked anti-rabbit and anti-mouse IgGs (CoWin Biotech, Beijing, China).