Absignal

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).

Whole cell and detergent-insoluble fractions were prepared as described previously [11 (link)]. Briefly, whole cell protein lysates and detergent-insoluble fractions were prepared in a modified RIPA buffer containing proteinase inhibitors and phosphatase inhibitors as described elsewhere [12 (link)]. Homogenates were spin down at 12,000 rpm at 4 °C for 20 min. Supernatants were collected for whole cell lysates and the pellets used as detergent-insoluble fractions. The pellets were dissolved in 0.1% SDS. Protein concentration of each samples was determined using BCA reagents (Thermo Scientific). Equivalent amounts of protein (20–80 μg) were loaded in 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred by blotting to polyvinylidene fluoride membranes. The blot was incubated with primary antibodies against human ZO-1, Occludin, Claudin-1, or GAPDH. After washing, the blot was incubated with HRP-conjugated secondary antibodies. The protein–antibody complexes were detected by Absignal (Abclone, Seoul, Korea) according to the manufacturer’s recommended protocol.