Syndecan 1

Primary antibodies were obtained from Abcam (SNAIL, N-cadherin; Cambridge, MA, USA), BD Transduction (E-cadherin; Franklin Lakes, NJ, USA), Santa Cruz Biotechnology (syndecan-1, Santa Cruz, CA, USA) and Contreras et al (24 (link)) (syndecan-2). Anti-rabbit secondary fluorescein-conjugated antibody, anti-mouse and anti-rabbit secondary peroxidase-conjugated antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

Protein extraction and blotting were performed as previously described [42 (link)]. After determination of protein concentration by a Protein Determination Kit (Cayman), equal amounts (50~100 μg) of protein samples were size fractionated using SDS-PAGE, electrotransferred onto PVDF membrane (Bio-Rad) and hybridized with antibodies. Antibodies used were for S1P₁ (TA311878, OriGene), Syndecan-1 (sc-390791, Santa Cruz), p-AKT (sc-33437, Santa Cruz), AKT (sc-8312, Santa Cruz), p-ERK1/2 (#9101, Cell Signaling), ERK1/2 (#4695, Cell Signaling), TGF-β1 (sc-52893, Santa Cruz), E-cadherin (sc-1500, Santa Cruz), Vimentin (sc-58899, Santa Cruz), Snail (#3879, Cell Signaling), and GAPDH (sc-137179, Santa Cruz). A 1:1000 dilution of those antibodies was used for detection. Densitometric quantification of bands was analyzed by the ImageJ software (version 1.50, NIH, USA).

HMVECs and TALL-104 cells were mixed (1:1) in culture for 12 h. Paraformaldehyde (4%) was added to the culture for 10 min at room temperature prior to cell lysate isolation, as described below.
For immunoblotting, cells were solubilized in ice-cold cell lysis buffer containing 6.52 mg/mL HEPES, 0.42 mg/mL NaF, 8.64 mg/mL NaCl, 0.2 mg/mL MgCl₂, 5% NP-40, and protease inhibitor cocktail (TOPSCIENCE, Shanghai, China). Proteins were fractionated by SDS-PAGE and transferred to PVDF membrane (Millipore, Burlington, MA, USA) and then blocked in 5% milk in PBST. Subsequently, the membrane was incubated overnight at 4 °C with one of a series of primary antibodies against mouse or rabbit PD-L1 (Cell Signaling, Danvers, MA, USA), PD-1 (Cell Signaling), VE-cad (Invitrogen, Waltham, MA, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Syndecan-1 (Santa Cruz Biotechnology), LEF1 (Cell Signaling), YKL-40 (our lab), CCR5 (Abcam, Cambridge, UK), AKT, pAKT (Cell Signaling), ERK, pERK, PI3K (Santa Cruz Biotechnology), Vimentin (Dako), SMa (Abcam), E-cad (Dako, MA, USA), GAPDH (Beyotime), or β-actin (Sigma-Aldrich). The membrane was then incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Beyotime). The specific chemiluminescence signal was detected using the ECL reagent (ThermoFisher Scientific).