Tnfsf9

First, the total cell protein was extracted with protein extract (Beyotime, China). Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000). Finally, the membrane was incubated with appropriate secondary antibodies for 1 h at room temperature. The band was quantitatively measured using the Zeiss LSM800 spectrometer (Carl Zeiss AG, Germany) [44 (link)].

Immunohistochemistry was performed on paraffin-embedded tissues. The specific experimental method is as described above [42 (link)]. TNFSF9 (1:100, ProteinTech) was used to incubate tissues at 4°C overnight. After the application of appropriate secondary antibodies, the labeled antigens were visualized using the standard 3,3′-diaminobenzidine (DAB) protocol. The slide is then back-dyed with hematoxylin. Immunoreactivity score (IRS) was used to evaluate tissue staining. The staining intensity is 0 to 3 points, 0 is no staining, 1 is low staining, 2 is medium staining, and 3 is high staining. The percentage of positive cells ranges from 0 to 4 points, 0 < 1%, 1 is 1 to 10%, 2 is 11 to 50%, 3 is 51 to 80%, and 4 is >80%. Multiplying the average intensity and percentage of positively stained cells produces an IRS, with a maximum score of 12 points. These data represent the average of the results obtained by the two scorers.