Clone 24e10

Cells were cultured on glass coverslips in 6-well plates to at least 80% confluence and fixed in ice-cold methanol for 20 min for both proximity ligation assays (PLA) E-cadherin/b-catenin and E-cadherin/p120. PLA was performed using Duolink Detection kit (Olink Bioscience, Sweden), according to the manufacturer’s instructions for Duolink Blocking solution and Detection protocol. Briefly, slides were blocked, incubated with antibodies directed against E-cadherin cytoplasmic domain (610,182, BD Biosciences or Clone 24E10, #3195, Cell Signaling, USA), b-catenin (C2206, Sigma-Aldrich, USA) and p120 (610,134, BD Biosciences, USA), followed by incubation with the secondary PLA probes (anti-mouse Minus and anti-rabbit Plus) conjugated to unique oligonucleotides. Amplification template oligonucleotides were hybridized to pairs of PLA probe and circularized by ligation. Rolling circle amplification was performed and detection of amplified DNA was possible by addition of complementary oligonucleotides labeled with Cy3 fluorophore. Coverslips were mounted on Vectashield with DAPI (Vector Laboratories, USA). Images were acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope (× 20 and × 40 objectives; Carl Zeiss, Germany) with an Axiocam HRm camera and processed with the Zeiss Axion Vision 4.8 software. Quantification of PLA signals was achieved using BlobFinder V3.2.42.

A549 cells for immunofluorescence staining were seeded in ibiTreat 8-chamber slides (Ibidi) and left to adhere overnight. Cells were then serum-starved for 16 h prior to pre-incubation with PF670462 (0.3 – 10 μM) for 30 min then TGF-β (100 pM) for 48 h. Cells were fixed in 10% neutral buffered formalin (Grale Scientific) for 15 min and non-specific binding sites were blocked by incubation with 5% normal goat serum/0.3% Triton X-100 in PBS for 1 h. E-Cadherin expression was detected using a rabbit monoclonal antibody (1:200, Clone 24E10; Cat#3195, Cell Signaling) followed by an AlexaFluor-488 conjugated anti-Rabbit F(ab′)₂ fragment secondary (1:500, Cat#4412, Cell Signaling). Specific binding was confirmed using an isotype control antibody (protein content matched to respective primary antibodies, Clone DA1E rabbit IgG; Cat#3900, Cell Signaling). Cell nuclei were then stained with DAPI. Cells were imaged using a Leica SP5 confocal microscope (Biological Optical Microscopy Platform, University of Melbourne). Cell morphology and immunofluorescent staining was quantified using the Operetta High Content Imaging System (Biological Optical Microscopy Platform, University of Melbourne).

SDS-PAGE and immunoblotting were performed as previously described by Geoghegan et al. [22 (link)]. Antibody information: TLR2 (AF2616-SP), Goat polyclonal (R&D Systems); TLR6 (NBP1-54336), Goat polyclonal (R&D Systems); IL-6Ra (MAB227), Mouse monoclonal IgG₁, Clone 17506 (R&D Systems); gp130 (MAB2281), Mouse monoclonal IgG_1, Clone 29104 (R&D Systems); Vimentin (5741), Rabbit monoclonal, Clone D21H3 XP^® (Cell Signaling); E-cadherin (3195), Rabbit monoclonal, Clone 24E10^® (Cell Signaling); N-cadherin (13116), Rabbit monoclonal, Clone D4R1H XP^® (Cell Signaling); β-catenin (8480), Rabbit monoclonal, Clone D10A8 XP^® (Cell Signaling); β-actin (4970), Rabbit monoclonal (Cell Signaling); GAPDH (CB1001) Rabbit monoclonal (Calbiochem); HRP conjugated anti-goat IgG (705-035-003) (Jackson Immunolabs); HRP conjugated anti-rabbit IgG (W401)(Promega).