Integrin β1

The antibodies used were obtained from the following suppliers: EGFR, EGFR-pY1068, ErbB3, ErbB3-pY1289, AKT, AKT-pS473, focal adhesion kinase (FAK), and FAK-pY397 were from Cell Signaling Technology (Danvers, MA, USA), Extracellular signal-related kinase (ERK) 1, ERK-pY204, and Integrin β1 were from Santa Cruz Biotechnology (Dallas, TX, USA), and α-tubulin was from Sigma-Aldrich.

HK-2 and Caki-1 cells used in this study were obtained from ATCC. Cell culture media and supplements, Dulbecco’s modified Eagle’s medium (DMEM) (Cat: MT10090CV; Corning), Macoy’s 5 A media, fetal Bovine serum (FBS) (F0926; Millipore Sigma), and antibiotic antimycotic solution (Cat: 30–004-Cl; Corning) were used. Primary antibodies used were integrin β1 (D2E5) Rabbit mAb (Cat: 9699; Cell Signaling); antibody for active integrin β1 12G10 (Cat: MAB2247; Millipore Sigma), integrin β1 (Cat: MABT821; Millipore Sigma), integrin β1, (Santa Cruz Biotechnology Cat: SC-374430), ACE2 monoclonal antibody (Cat: MA5–32307; Thermo Fisher), GAPDH (Cat: 5174; Cell Signaling). Anti His antibody (Cat: SC53073, Santa Cruz Biotechnology). Spike protein (Cat: 230–20407–200, Ray Biotech) and the Control protein (Cat: 230–20001, Ray Biotech).

Cells were fixed with 4% paraformaldehyde for 10  min and permeabilised in 0.1% Triton X-100 for 10  min. Cells were blocked in 1% BSA for 1  hr before incubation with primary antibodies – pS19-MLC (Cell Signaling #3671 L), myosin MHC IIa (Covance PRB-440P), fibronectin (Sigma F3648), β-catenin (Santa Cruz sc7963), integrin β1 (Santa Cruz sc13590), and integrin β3 (Abcam, ab179473) at 4°C overnight. After incubation, the appropriate fluorescence-conjugated secondary antibodies for 1 hr, cells were washed with PBS. Images were acquired with an inverted Zeiss LSM780 at a magnification of ×20 and ×63. For quantification of the pMLC staining, regions of interest were drawn around equal numbers of ‘free boundary zones’ of A431 cells in clusters and cell-cell contact zones, and the mean fluorescent intensity was measured. The values were then normalised to the mean of all the boundary and contact zones for WT A431 cells. Staining of frozen human tissue sections was performed in a similar manner, except that fixation and permeabilisation times were doubled, and 5% BSA was used as a block.

The antibodies c-Myc, MAPK (Erk1/2), Phospho - MAPK (Erk1/2), Stat3, Phospho - Stat 3, AKT and Phospho - AKT were obtained from cell signaling technology, MA, USA. The antibodies P63, AR, Integrin β1, Bcl-2, GAPDH, Wnt-1(G-19) and TGFβ1 were derived from Santa Cruz Biotechnology, CA, USA. The antibodies CD133, CK8, E Cadherin, N Cadherin, and Snail were derived from Abcom, MA, USA. The antibody CK5 was obtained from Covance, CA, USA. The antibody CD44 was derived from eBioscience, CA, USA. The antibody Vimentin was derived from Fisher Scientific, MA, USA. The antibody Ki67 was obtained from Novocastra, Newcastle, United Kingdom.

AGS wt and AGSΔcttn cells intended for SDS-PAGE were harvested with hot (95 °C) SDS-buffer, followed by separation of the proteins in the lysates, according to size via 6–8% polyacrylamide gels. The proteins were then transferred onto a PVDF membrane for Western blotting and probed with antibodies [69 (link)]. Prior to probing, the PVDF membranes were blocked with either 3% BSA or 5% non-fat dry milk in TBST, depending on the primary antibody and manufacturers instructions [70 (link)]. Primary antibodies used were specific against cortactin (Merck-Millipore, Darmstadt, Germany #05-180), GAPDH (Santa Cruz, Heidelberg, Germany, #SC-47724), CagA (Austral Biologicals, San Ramon, CA, USA, #HPP-5003-9), CagE [63 (link)], VacA [34 (link)], FlaA [71 (link)], RPTP-α (Santa Cruz, #SC-28907), integrin-β1 (Santa Cruz, #SC-H96), full-length caspase-3 (Cell Signaling Technology, Frankfurt, Germany, #9668) and active caspase-3 (Cell Signaling Technology, #9664). Secondary antibodies detected either mouse (Invitrogen, Darmstadt, Germany, #31446) or rabbit (Invitrogen, #31460) primary antibodies and were coupled to horseradish peroxidase. Development of Western blots was performed as previously described [72 (link)].

Rapid-immunoprecipitation assay (RIPA) buffer, blocking solution, and protease inhibitor were purchased from GenDepot (Barker, TX, USA). β-Tubulin and RUNX2 antibodies were purchased from Proteintech (Rosemont, IL, USA), and integrin α5, integrin αV, N-cadherin, E-cadherin, and integrin β1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Total p38, p–p38, total ERK1/2, and p–ERK1/2 antibodies were purchased from Cell Signaling Technology (Billerica, MA, USA). After primary antibody incubation, membranes were washed with PBST and incubated with horseradish conjugated secondary antibodies for 1 h at room temperature. Signal was visualized by enhanced chemiluminescence (ECL) using Azure300 (Azure Biosystems, Dublin, CA, USA), and intensities were quantified using a computing densitometry program from Image Studio Lite (LI-COR, Lincoln, NE, USA).