Ltbp1

The expression of protein in 152 ESCC tissues was detected using an immunoperoxidase method. IHC staining was performed according to standard protocols as previously described [24 (link)]. The slides were incubated in primary antibody (LTBP1(1:1000, #26855; Proteintech), FN1 (1:2000, #66042; Proteintech), E-Cadherin (1:200, #3195; Cell Signaling) and TGFβ1(1:60, #ab92486, Abcam) and followed by treatment with secondary antibody and a DAB staining kit (Gene Tech #GK500705). IHC intensity for each tissue section was evaluated independently by 2 experienced pathologists who were blinded to patients’ clinical data.
Cells were incubated in primary antibodies α-SMA (1:100, #14395; Proteintech) and FN1 (1:100, #66042; Proteintech). The cells were washed with PBS for three times and followed by treatment with CoraLite488 and CoraLite594 labeled IgG (Proteintech) for an hour. After washing the cells for four times, cells were stained by DAPI (Sigma).

The proteins were separated by 10% SDS-PAGE and transferred to PVDF Membranes (Millipore). The membranes were blocked in 5% BSA for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4 °C, GAPDH(1:5000, #10494; Proteintech), β-tubulin(1:2000, #10068; Proteintech), LTBP1(1:500, #26855; Proteintech) and FN1(1:500, #66042; Proteintech), PLOD2 (1:500, #21214; Proteintech), THBS1(1:500, #bs-2715R; Bioss), RCN3(1:500, #ab204178; abcam), α-SMA (1:1000, #14395; Proteintech), Vimentin (1:1000, #5741; Cell Signaling), N-Cadherin (1:1000,#13116; Cell Signaling), E-Cadherin (1:1000, #3195; Cell Signaling), BCL2 (1:1000, #4223; Cell Signaling), BAX (1:1000, #5023; Cell Signaling). The membranes were washed thrice with TBST and incubated with HRP-conjugated secondary antibody (1:10000) 1 h at room temperature. The membranes were washed thrice with TBST and visualized using ECL (Thermo Scientific). The relative density was quantified by ImageJ.