Ecl kit

Whole-cell protein extracts were prepared as previously described,^{22 (link)} and proteins were detected by immunoblotting with the primary antibody from cell signalling (E-Cadherin, MST1, YAP/TAZ, P-Ser127YAP, GAPDH, vimentin), diluted to 1:1000 in Tween (0.1%)–tris-buffered saline buffer and horseradish peroxidase-conjugated secondary antibody, and then revealed by enhanced chemiluminescence using the ECL kit (Promega™). Densitometry results of western blot were analysed with ImageJ software. The signal intensity of each band was normalised with GAPDH densitometry values.

Western blot analysis of the PPV-C CP was carried out as described previously [30 (link)]. Leaf samples were homogenized in the 2× loading buffer using 1/15 ratio (w/v) and heated at 96 °C for 7 min. Proteins were resolved by 10% (w/v) SDS-PAGE and transferred to Immobilon-P membrane (Millipore, Burlington, MA, USA). Membrane was blocked for 1 h at room temperature with 5% (w/v) skim milk powder (Sigma-Aldrich, St. Louis, MO, USA), diluted in PBS, and probed with the antibody 5B at the 1/10,000 dilution. HRP-labeled anti-mouse IgG (W4021, Promega, Madison, WI, USA) was used as the secondary antibody (1/20,000 dilution). PBS with 0.1% (v/v) Tween 20 was used for membrane washing and dilution of immunospecific reagents. Detection was performed employing ECL kit (Promega).

The antibodies used for IHC were the same as those used for WB. Whole-cell protein extracts were analyzed by WB, as previously described [25 (link)], with proteins detected with an enhanced chemiluminescence technique using the ECL kit™ (Promega).

Authenticated HEK293 cells were purchased from ATCC and tested for mycoplasma by PCR. HEK293 cells were grown in six well plates and transfected with 4 µg of each DNA with lipofectamine 2000 (Invitrogen) for 6 hr according to manufacturers instructions.
For immunohistochemistry, cells were fixed 48 hr after transfection in 4% paraformaldehyde in PBS for 20 min at room temperature, washed with PBS, and permeabilized in 0.2% Triton X-100 in PBS for 5 min at room temperature. The protocol was followed as in Supp. Materials and methods.
For immunoprecipitation, cells were grown for 24 hr after transfection and then proteins were extracted following standard methods (Supp. Materials and methods). The eluate from antibody beads (30 µl) was loaded in 10% polyacrylamide gels and proteins were detected by Western blots (standard conditions) using anti-myc (1/20,000, SC-40, SCBT), anti-HA (1/10,000, 3F10, Roche) and anti-β-catenin (1/8000, Sigma, C7207), to detect the co-immunoprecipitated proteins. Antibodies used on Western blots in Figure 3—figure supplement 1B are anti human Tcf7l2 (N-20, SCBT) and anti-gamma tubulin (T9026, Sigma) HRP coupled secondary antibodies (1/2,000, sigma) were used and blots were developed using an ECL kit (Promega).