Antibodies against β catenin

The protein levels of β-catenin and α-SMA in ECSC and NESC were evaluated by western blot analysis. Antibodies against β-catenin (Cell Signaling, MA, USA), α-SMA (Gene Tex, Irvine, California, USA), and GAPDH (FUJIFILM Wako Chemical Corporation, Osaka, Japan) were used. Total protein from ECSC cells that were treated with ICG-001 (20 µM) or C-82 (2 µM) for 48 hours was extracted. The expression of each protein relative to the GAPDH protein in ECSC and NESC was analyzed using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA). The data were presented as the percentage of the values obtained for ECSC compared with NESC.

Cholesterol, POPC, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were purchased from Avanti Polar Lipids. Human Wnt3a protein was purchased from R&D Systems (Cat no. 5036-WN-010; lot no. RSK9221052). siRNA for human TSC2 was purchased from Integrated DNA Technologies (Ref. no. 311363879) and the transfection reagent JetPRIME was from Polyplus-transfection. Antibodies against β-catenin (Cat no. 9587S), active (nonphospho) β-catenin (Cat no. 8814S), Tuberin/TSC2 (Cat no. 4308S), p70 S6 kinase (S6K) (Cat no. 2708S), phospho-p70 S6K (pS6K) (Cat no. 9205S), and glyceraldehyde 3-phosphate dehydrogenase (Cat no. 5174S) were purchase from Cell Signaling Technologies. Anti-Rabbit IgG, HRP-linked antibody (Cat no. 7074S) and anti-Mouse IgG as well as HRP-linked antibody (Cat no. 7076S) were also from Cell Signaling Technologies. HEK293T and HeLa cells were purchased from ATCC. Chemicals for Cholesterol oxidase assay activity, including 4-aminoantipyrine (Cat no. A4382-10G), phenol (Cat no. P1037-25G), and peroxidase from horseradish (Cat no. P8375-1KU) were purchased from Millipore Sigma.

Antibodies against β-catenin, Akt, and PI3K were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against TGF-β1 and α-SMA were from Abcam (UK). The antibody against GAPDH was from Hangzhou Xianzhi Biological Technology (Zhejiang, CN). Anti-β-actin antibody was from Sigma. Anti-mouse IgG peroxidase-linked whole antibody and anti-rabbit IgG peroxidase-linked species-specific whole antibody were from Beyotime Institute of Biotechnology (Jiangsu, CN).

Western blot analysis was performed according to standard procedures. Equal amounts of protein (40 μg) from each sample were separated on SDS-PAGE precast gels (Invitrogen Life Technologies) and transferred onto nitrocellulose membranes (Invitrogen Life Technologies). After blocking with 5 % skim milk, the membranes were blotted with anti-bodies against β-catenin, c-Myc, Bax, Bcl-2, GADPH (all from Cell Signaling Technology, USA) or Dvl-1(Santa Cruz, USA) by using the Western Breeze chromogenic immunodetection system (Invitrogen Life Technologies),the images were captured and quantified with Image Lab 4.0 software (Bio-Rad Laboratories, USA).

For the detection of SggEsxA, TX20005 and TX20005Δesx were grown in BHI at 37°C with shaking for ~18 hours. Cultures were centrifuged, supernatants collected, filtered through a 0.2 μm filter, and concentrated using the Pierce™ Protein Concentrator PES, 3K Molecular Weight Cutoff. Bacterial pellets were washed and resuspended in PBS. Supernatants and bacterial suspensions were boiled in SDS loading dye and subjected to SDS-PAGE. Membranes were probed with anti-EsxA rabbit serum (1:100) (Pacific Immunological) and anti-FtsZ (1:250) antibodies (Abbexa, cat no. abx319936), followed by incubation with secondary antibodies conjugated to horse radish peroxidase (HRP) (1:3000). Blots were washed and immersed in Clarity Max ECL (Bio-rad). Images were acquired using a Fluorchem M chemiluminescent imager (Protein Simple).
Detection of β-catenin and PCNA was carried out as described previously [12 (link)]. Briefly, total cell lysates from HT29 cells cultured in the presence or absence of bacteria or CS were subjected to SDS-PAGE, transferred, and probed with antibodies against β-catenin (1:1000), PCNA (1:1000), and β-actin (1:1000) (Cell Signaling Technology), followed by incubation with HRP-conjugated secondary antibodies (1:3000). Band intensities were measured using ImageJ and normalized to that of β-actin.