Ab49254

Cells were washed with PBS and lysed with SDS lysis buffer. Frozen tissues were lysed using T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific). Protein concentrations were determined using the Bradford assay (Bio-Rad). Equal amounts of protein from each sample were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore). Immunoblots were blocked by incubation in 5% skim milk in TBS-T (0.1% Tween 20) for 1 h at 25 °C. Membranes were incubated with the following primary antibodies: anti-TINAGL1 (1:1000; 12077-1-AP, Proteintech), anti-pFAK (1:1000; #3283; Cell Signaling Technology), anti-FAK (1:1000; #3285; Cell Signaling Technology), β-actin (1:10,000; sc-47778, Santa Cruz Biotechnology), anti-Twist (1:1000; ab49254, Abcam), anti-E-cadherin (1:500; #13-1700; Invitrogen), anti-N-cadherin (1:1000, ab18203, Abcam), anti-integrin β1 (1:1000; ab30394, Abcam), anti-integrin αv (1:1000; #4711, Cell Signaling Technology), anti-integrin α5 (1:1000; #4705, Cell Signaling Technology), and anti-GAPDH antibodies (1:20,000; Abc-1001, AbClon), followed by their corresponding HRP-conjugated secondary antibodies, anti-mouse (1:5000: #115-035-003, Jackson ImmunoResearch Labs) and anti-rabbit antibodies (1:4000; #Abc-5003, AbClon). Proteins were detected using AbSignal (Abclone).

Immunoblots were performed with samples containing total protein (40 μg) and 12% NuPage Bis-Tris or 7% Tris-Acetat polyacrylamide gels (LifeTechnologies). The membranes were probed with rabbit polyclonal anti-PER1 (1:500; Abcam ab136451), rabbit polyclonal anti PER2 (1:250; ab180655), rabbit monoclonal anti-PER3 (1:2500; ab177482), rabbit polyclonal anti-DBP (1:400; ab22824), rabbit polyclonal anti-Twist1 (1:400; ab49254) and rabbit polyclonal anti-TWIST2 antibodies (1μg/ml; ab66031). The secondary antibody was a horseradish peroxidase-linked goat anti-rabbit IgG (various concentrations; ab97051), and it was detected by the chemiluminescence technique using the Immobilon Western ECL system (Millipore). For a loading control, we used a mouse monoclonal anti-p84 antibody (nuclear matrix protein 84; 1:2000; Abcam ab487). The secondary antibody was a horseradish peroxidase-linked goat anti-mouse IgG (1:5000; Abcam; ab20043). Densitometric analysis was performed using the ImageJ software and the intensity of the control lanes were set as 1.

We extracted total proteins from human GC tissues and cell lines using the RIPA lysis buffer (AS1004, ASPEN, Wuhan). Then, equal amounts of the protein lysates were separated on a 10% SDS PAGE. The resolved proteins were transferred onto PVDF membranes (IPVH00010, Millipore) and blocked with 5% non-fat milk in TBST for 2 h at room temperature. Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti- SERPINH1 (ab109117, Abcam), anti-Wnt2 (sc-514382, SANTA CRUZ), anti-β-Catenin (17565-1-AP, SanYing, Wuhan), anti-p-GSK3β (#5558, CST), anti-GSK3β (#12456, CST), anti-NFκB p65 (#8242, CST), anti-Snail1 (13099-1-AP, SanYing, Wuhan), anti-Slug (ab106077, Abcam), anti-TWIST (ab49254, Abcam), anti-MMP2(ab37150, Abcam), anti-MMP9 (ab76003, Abcam), anti-E-Cadherin (#3195, CST), anti-N-Cadherin (#4061, CST). Subsequently, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (AS1107, ASPEN). The blots were developed using the enhanced chemiluminescence system (AS1059, ASPEN) and the protein bands were imaged.

Whole cell protein (25~60 μg) extracted by RIPA lysis buffer from each sample was loaded into 8% or 10% polyacrylamide gel for electrophoresis as previously described (Cheng et al., 2018 (link)) followed by membrane transfer carried out at 300 mA for 1.5~2.5 h. Membranes were blocked with 5% non-fat milk in Tris-buffered saline solution (pH 7.4) containing 0.05% Tween-20 and incubated with primary antibodies overnight. The following primary antibodies were used. β-actin (20536-1-AP, Proteintech, China), E-cadherin (24E10, CST, USA), N-cadherin (ab18203, abcam, USA), Estrogen receptor α (ab32063, abcam, USA), Twist (ab49254, abcam, USA), and Snail (L70G2, CST, USA). After incubation with secondary antibodies (A0208, Beyotime, China), the membranes were treated with reagents from the ECL kits (P90719, Millipore, USA) and exposed to X-ray film (Kodak, USA) in darkness.

Western blotting staining and immunohistochemical (fluorescence) staining were performed as described previously 17, 19. The primary antibodies used in this study were Snail (1:1,000 dilution; MABE167; Merck, Darmstadt, Germany), E‐cadherin (1:1,000 dilution; #3195S; Cell Signaling Technology), PCAF (1:1,000 dilution; #3378S; Cell Signaling Technology), vimentin (1:2,000 dilution; GTX100619; GeneTex), β‐actin (1:10,000 dilution; #4967L; Cell Signaling Technology), HA (1:1,000 dilution; #3724S; Cell Signaling Technology), GFP (1:500 dilution; SC‐9996; Santa Cruz Biotechnology), ISX (1:200 dilution; sc‐398934; Santa Cruz Biotechnology), TWIST1 (1:200 dilution; ab49254; Abcam), BRD4 (1:1,000 dilution; #13440S; Cell Signaling Technology), acetylated lysine (1:500 dilution; #9441S Cell Signaling Technology), fibronectin (1:500 dilution; GTX112794; GeneTex), mCherry (1:500 dilution; GTX128508; GeneTex), Slug (1:1,000 dilution; GTX128796; GeneTex), VEGF (1:200 dilution; sc‐7269; Santa Cruz Biotechnology), and N‐cadherin (1:1,000 dilution; GTX127345; GeneTex). FITC‐conjugated anti‐rabbit IgG, rhodamine‐conjugated anti‐mouse IgG, and alkaline phosphatase‐conjugated anti‐rabbit IgG antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were also used. All experiments were repeated at least three times.