Wlp1512

Immunocytochemical staining was performed on the coverslips obtained from each of the experimental groups, with the use of rabbit polyclonal antibodies against pERK (1:300, WLP1512, Wanleibio, Shenyang, China). Briefly, the coverslips were washed three times with PBS, permeabilized with 0.3% Triton X-100 for 15 min at room temperature, incubated with 3% H₂O₂ for 10 min, blocked by normal goat serum for 30 min at 37 °C, and then with the appropriately diluted first antibody at 4 °C overnight in a humid chamber. The color reaction was carried out using 3,30-diaminobenzidine tetrahydrochloride (DAB). For immunofluorescence labeling, the THJ-16T or THJ-21T-bearing coverslips of the experimental groups were rinsed three times with PBS, permeabilized with 0.3% Triton X-100 for 15 min, blocked with 10% goat serum in PBS for 30 min at 37 °C, then incubated with primary antibody pSTAT3 (1:300, ab76315, abcam, Cambridge, UK) overnight at 4 °C, followed by Coralite488-conjugated goat anti-rabbit IgG (1:500, SA00013-2, Proteintech, Wuhan, China) at 37 °C for 60 min in the dark. Nuclei were labeled with Hoechst 33342 (C1025, Beyotime Biotech, Shanghai, China), and images captured by fluorescence microscope (Nikon, ECLIPSE Ni-U).

Cell lines were washed twice with phosphate-buffered saline (PBS) before being lysed with radio-immunoprecipitation assay buffer (Wanleibio). Protein concentrations were determined using the bicinchoninic acid protein assay kit (Wanleibio) according to the manufacturer’s protocol. Equal amounts of protein lysate (30 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After being blocked with 5% skim milk, the membranes were incubated with primary antibodies (1:1,000) against SPRED1 (Abcam, Cambridge, UK, no. 77079), (1:400) against p-ERK (Wanleibio, WLP1512), (1:400) against ERK (Wanleibio, WL01864), (1:500) against cleaved-caspase-3 antibody (Wanleibio, WL01992), (1:500) against p-P53 (Wanleibio, WL03214), (1:1,000) against Bax (Wanleibio, WL01637), (1:1,000) against Bcl2 (Wanleibio, WL01556), and β-actin (Wanleibio, WL01845) overnight at 4°C. The same membrane was washed with Tris-buffered saline with Tween 20 and reblotted with peroxidase-conjugated goat anti-rabbit (1:5,000 Wanleibio, WLA023) for 45 min at 37°C. For quantitative analysis, the bands were selected and quantified using Gel-Pro-Analyzer Software, and the data were transformed and normalized to β-actin.