Ccl22

Proteins of HUVECs or fresh tissues were prepared for western blot analysis. Protein concentration of each sample was quantified with the BCA protein assay (Beyotime Institute of Biotechnology, China). Then, equal amounts of proteins were separated by electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% milk in Tris-buffered saline and 0.2% Tween at room temperature for 1 h and then incubated overnight at 4 °C with the following specific primary antibodies: GAPDH (1:1000, Cell Signaling Technology), ERK (1:1000, Cell Signaling Technology), p-ERK (1:1000, Cell Signaling Technology), p38 (1:500, Abcam), p-p38 (1:1000, Cell Signaling Technology), JNK (1:500, Santa Cruz Biotechnology), p-JNK (1:500, Santa Cruz Biotechnology), CCL17 (1:1000, Abcam), and CCL22 (1:1000, Abcam). The blots were washed three times with Tris-buffered saline and 0.2% Tween and incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 45–60 min at room temperature. The expression signals were detected with an enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology, China) after the membranes were washed with TBST (10 min × 3).

Total protein of tumor tissue samples was extracted and denatured for electrophoresis with 16.5% tricine gel (ShareBio, China). The separated proteins were transferred to PVDF membranes and then hybridized with primary and HRP-conjugated secondary antibodies (CST, America), respectively. Target protein bands were visualized with a gel imaging system (Tanon, China). The primary antibodies for IL6 (1:1000), CCL3 (1:1000), CCL4 (1:5000), CCL22 (1:3000), CXCL11 (1:2000) were purchased from Abcam (Cambridge, England); CCL2 (1:1000), CCL20 (1:500), CXCL1 (1:500), CXCL16 (1:500) were purchased from Affinity Biosciences (Jiangsu, China); CCL27 (1:500) was purchased from ABclonal Technology (Boston, USA).