Phosphatase inhibitor

The activity of superoxide dismutase (SOD), catalase (CAT), and peroxide (POD) in workers after injection with ds301A1, ds303A1, ds306A1 as the treatment groups and dsGFP as the control group (CK) separate at 12 or 24 h was measured with the SOD, CAT, or POD enzyme-linked immunosorbent assay (ELISA) kit (MIbio, China). Samples were homogenized in cell lysis buffer containing 10 mM phosphatase inhibitor (CWBIO, Beijing, China). The supernatants were recovered by centrifugation at 10,000g and 4°C for 10 min. Protein concentrations were quantified by a BCA Protein Assay Kit (Beyotime, Beijing, China). Next, ELISA analyses were carried out as described by Meng et al. (2018) (link) to evaluate the activity of antioxidant enzymes. The optical densities of each well were measured with a Multiskan (BioTek Instruments, USA) at a wavelength of 450 nm. The activities of SOD, CAT, and POD were calculated according to the calibration curve, and the calibration curves are generated with the immunosorbent assay kit (ELISA) according to the manufacturer’s instructions. Each sample was run in three biological and three technological replicates. A one-way ANOVA was performed to analyze the differences between the control and treatment groups.

The tumor tissues and cells were lysed using the RIPA lysis buffer containing PMSF and a phosphatase inhibitor (CWbiotech, Beijing, China). They were centrifuged at 12,000×g and 4 °C for 5 min to collect the total cell lysates. Afterward, the protein content in the supernatants was determined using the BCA Protein Assay Kit (Solarbio, Beijing, China). Then, 20 µg of the corresponding protein extracts were used, separated on 10% SDS-PAGE gels, and transferred onto a PVDF membrane (GE Healthcare Life). After washing with tris-buffered saline (TBST), the membranes were blocked with 5% fat-free milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against JMJD2A (1:1000), STMN1 (1:1000) and β3-Tubulin (1:1000). On the following day, the membranes were washed three times for 10 min each with TBST, then incubated at room temperature for 2 h with goat and anti-rabbit IgG secondary antibodies (Cell Signaling Technology, Boston, USA). Immunoreactive proteins were visualized using the ECL chemiluminescence kit (Solarbio, Beijing, China). GAPDH was used as the internal reference. Band intensities were determined using the Chemi Capture (CLiNX, Shanghai, China) software.

Cell samples were collected and quickly incubated with cold RIPA‐strong lysis buffer (Beyotime Biotechnology) which contains 1% protease inhibitor cocktail (Cwbiotech) and 1% phosphatase inhibitor (Cwbiotech) over 30 min. The lysis sample was then centrifuged at 12,500 rpm at 4°C for 30 min. The supernatant was collected, and a BCA (Thermo Fisher Scientific) assay was used for protein quantitation. A total of 30 μg of protein sample was added to each lane of a sodium dodecyl sulphate–polyacrylamide gel. Proteins were transferred to a polyvinylidene fluoride membrane (0.2 μm; Millipore) after electrophoresis. The membrane was then blocked with 5% skimmed milk at room temperature for 1 h and incubated with the indicated primary antibodies overnight at 4°C. All primary antibodies used in this research were diluted in 1:1000. Later, the membranes were washed with PBST buffer and incubated with secondary antibody for 2 h at room temperature. The secondary antibodies were diluted in 1:10,000. Finally, the targeted protein band was illuminated with ECL Western Blotting Substrate (Millipore).