Anti fibronectin

Cell lysates were collected in RIPA lysis buffer (1% Triton-X-100, 20 mM Tris, pH 7.5, 137 mM NaCl, 1 mM EGTA, 10% glycerol, 1.5 mM MgCl₂, and protease inhibitor mixture and phosphatase inhibitors; latter 2 were from Roche). Lysates were sonicated and centrifuged at 4 °C. Per lane, whole-cell lysate was separated on 12% SDS-acrylamide gels and transferred on Immobilon PVDF membranes (Millipore). The membranes were probed with primary antibodies overnight at 4 °C and incubated for 1 h with secondary peroxidase-conjugated antibodies (CST). Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology) and detected by the ChemiDoc XRS gel documentation system (Bio-rad). Antibodies include anti-Ube2v1 (Abcam, monoclonal ab151725), anti-E-cadherin (Santa Cruz, monoclonal sc-21791), anti-N-cadherin (Boster, polyclonal BA0673), anti-Fibronectin (Boster, polyclonal BA1771), anti-Vimentin (Abcam, monoclonal, ab8978), anti-β-Catenin (Cell Signaling Technology, monoclonal #8480), anti-Twist1 (Proteintech, 25465), anti-Snai1 (Bioss, bs-2441R), anti-LC3B (CST, monoclonal 3868), anti-SQSTM1/p62 (CST, polyclonal 5114), anti-H4K16ac (Immunoway, polyclonal YM3317), anti-Beclin1 (Boster, polyclonal PB0014), anti-histone H3 (Abcam, polyclonal ab1791), and anti-Sirt1 (Santa Cruz, monoclonal sc-74504).

Paraffin-embedded slides were incubated with primary antibodies: anti-Ube2v1 (Abcam, polyclonal ab88679), anti-E-cadherin (Dako, monoclonal M3612), anti-Fibronectin (Boster, polyclonal BA1771), anti-Vimentin (Abcam, monoclonal, ab8978), anti-β-catenin (Cell Signaling Technology, monoclonal #8480), anti-SQSTM1/p62 (CST, polyclonal 5114), and anti-Beclin1 (Boster, polyclonal PB0014). Staining was done on a SPlink Detection Kit (SP-9000). We quantified staining intensity and percentage of stained cells. Positive tumor cells were quantified by two independent observers. The staining intensity was scored on a scale of 0–3 as negative (0), weak (1), medium (2), or strong (3). The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tumor area, was scored on a scale of 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). An overall protein expression score (overall score range, 0–12) was calculated by multiplying the intensity and extent positively scores.