Mouse anti human flag

Rhodamine-phallotoxin was purchased from Molecular Probes (Eugene, OR, USA). Mouse anti-human FHL2 used for western blot were a product of the MBL international incorporation (11–134, MBL International Incorporation, Woburn, Japan). Rabbit anti-human FHL2 antibody was used for IHC and was purchased from Abcam (Cambridge, UK). Mouse anti-FOXK1 (G-4), Vimentin (E-5), E-Cadherin (H-108), GAPDH (G-9) and bovine anti-mouse IgG-TR and goat anti-rabbit IgG-FITC were purchased from Santa Cruz (Santa Cruz, CA, USA). Mouse anti-human Flag were a product of Sigma (St Louis, MO, USA). Goat anti-rabbit immunoglobulins/HRP, rabbit anti-mouse immunoglobulins/HRP, normal mouse and rabbit IgG were all products of Dako (Carpinteria, CA, USA).
Human colon cancer cell lines SW480, Caco2 and SW620 were all maintained in our laboratory as previously described.^{26 (link), 27 (link)} The cells were cultured in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin in a humidified incubator at 37 °C with an atmosphere of 5% CO₂.

Cell samples were lysed in RIPA buffer (Thermo Fisher Scientific, #89900) and protein concentrations were quantified using the Pierce BCA kit (Thermo Fisher Scientific, #23225). Denatured proteins (10–20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Afterward, the membranes were blocked with 5% fat-free milk (BD Biosciences, #232100) in Tris buffered saline containing Tween 20 (TBST) and incubated with rabbit anti-human MYCN (1:1000; Cell Signaling Technology, #84406), mouse-anti-human Flag (1:1000; Sigma, #F1804), or rabbit-anti-human β-tubulin (1:5000; Abcam, #ab6046) primary antibodies overnight. The membranes were then incubated with HRP-conjugated goat anti-rabbit or mouse IgG (0.2 μg/ml; Pierce, #31460 or #31430). A luminescent image analyzer (Fujifilm, LAS-4000) was used to visualize the bands after an incubation with enhanced chemiluminescence reagents (Millipore, WBKLS0500).