Gtx128508

The following antibodies were used for staining of the imaginal discs: mouse anti-myc 1:500 (Cell Signaling Technology, Frankfurt, Germany), guinea pig anti-H 1:250 [96 (link)], rabbit anti-mCherry 1:500 (GTX128508, GeneTex, obtained from Biozol, Eching, Germany); secondaries coupled to FITC or Cy3 were used at 1:200 (Jackson Immuno-Research, obtained from Dianova, Hamburg, Germany). Imaginal discs within the same plane were chosen, and five 1 µm thick sections taken at identical confocal settings using a Zeiss Axioskop coupled with a BioRad MRC1024 confocal microscope, using LaserSharp 2000 software (Carl Zeiss, Jena, Germany). For the quantification of signal intensity, sections were stacked, and the mean gray value of the entire disc recorded using ImageJ. Measurements of ten discs were sampled, the mean and SD (standard deviation) are indicated. Statistical analyses were performed by ANOVA for multiple comparisons, using a two-tailed Dunnett’s approach.

Coverslips were fixed with 4% PFA w/v and 15% w/v sucrose solution in PBS for 20 min at room temperature and slides were washed three times in PBS on a rocking plate. Blocking buffer consisting of 3% w/v BSA and 0.1% v/v Triton X in PBS was added for 1 h and slides were washed again. Primary antibodies anti‐MYC (Proteintech, 60003‐2‐Ig, 1:10 dilution factor) and anti‐mCherry (GeneTex, GTX128508, 1:100 dilution factor) were added in PBS with 0.1% v/v Triton X overnight at 4°C. The next day, slides were washed three times, and secondary antibodies were added at 1:500 dilution factor in PBS for 1 h at room temperature. Finally, slides were washed and Fluosave and glass coverslips (VWR International) were added. Slides were stored in the dark at 4°C until imaging was performed using an Axioplan2 microscope (Zeiss) and Axiovision software (Zeiss).

Western blotting staining and immunohistochemical (fluorescence) staining were performed as described previously 17, 19. The primary antibodies used in this study were Snail (1:1,000 dilution; MABE167; Merck, Darmstadt, Germany), E‐cadherin (1:1,000 dilution; #3195S; Cell Signaling Technology), PCAF (1:1,000 dilution; #3378S; Cell Signaling Technology), vimentin (1:2,000 dilution; GTX100619; GeneTex), β‐actin (1:10,000 dilution; #4967L; Cell Signaling Technology), HA (1:1,000 dilution; #3724S; Cell Signaling Technology), GFP (1:500 dilution; SC‐9996; Santa Cruz Biotechnology), ISX (1:200 dilution; sc‐398934; Santa Cruz Biotechnology), TWIST1 (1:200 dilution; ab49254; Abcam), BRD4 (1:1,000 dilution; #13440S; Cell Signaling Technology), acetylated lysine (1:500 dilution; #9441S Cell Signaling Technology), fibronectin (1:500 dilution; GTX112794; GeneTex), mCherry (1:500 dilution; GTX128508; GeneTex), Slug (1:1,000 dilution; GTX128796; GeneTex), VEGF (1:200 dilution; sc‐7269; Santa Cruz Biotechnology), and N‐cadherin (1:1,000 dilution; GTX127345; GeneTex). FITC‐conjugated anti‐rabbit IgG, rhodamine‐conjugated anti‐mouse IgG, and alkaline phosphatase‐conjugated anti‐rabbit IgG antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were also used. All experiments were repeated at least three times.