Sc 10436

ChIP assay was performed by using the Upstate-ChIP Assay Kit (Lake Placid, NY, USA) following the manufacturer's instructions. In brief, DNA from MCF-7 and MCF-7/ADR cells was submitted to immunoprecipitation with 4 μg of normal goat IgG or anti-Slug (sc-10436, Santa Cruz Biotechnology). Then, ChIP-enriched DNA was analyzed by qRT-PCR using the ABI PRISM 7900HT sequence detection system and SYBR green master mix. Primers used were: GAPDH, 5'-agcgcaggcctcaagacctt-'3 (forward) and 5'-aagaagatgcggctgactgt-3' (reverse), and MMP1 promoter, 5’-ttttaatgggcaggagatgc-3’ and 5'-ggatgatgaaaaggctggaa-3' (reverse).

Five 4-µm cross-sections, mounted on previously silanized slides, were
used to show the expression of 8-OHdG, MMP-2 and MMP-9. The slides were then
deparaffinized, cleared, hydrated and washed in running water. Then, endogenous
peroxidase activity was blocked with 0.3% hydrogen peroxide, protein blocking
was performed with 0.3% skim milk diluted in PBS and the slides were incubated
overnight with anti-8-OHdG (SC66036 Santa Cruz® Biotechnology, CA, USA)
primary antibody, titrated 1:100, and MMP2/72KDa and MMP9/KDa (SC-10436 Santa
Cruz® Biotechnology, CA, USA; SC-6840 Santa Cruz^®Biotechnology, CA, USA), titrated 1:150 in PBS-BSA 0.1%. All slides were then
placed in a humid chamber at 4°C overnight. The material was washed with PBS
buffer and incubated with biotinylated secondary antibody. For revelation, the
3-3´diaminobenzidine chromogenic substrate was used at a ratio of 0.06 g per 100
mL of PBS, and 1 mL of 20-volume H₂O₂ for five minutes at
37°C and counter-stained with Mayer’s hematoxylin for 3 minutes. Finally, the
slides were mounted with cover slips and entellan® for analysis under
light microscopy.

Tissues and cells were lysed in 10 volumes (w/v) of lysis buffer. After centrifugation, the supernatant was collected and quantified. The same amount of total protein was separated by 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was then incubated overnight at 4°C with primary goat anti-human polyclonal antibody Btbd7 (ab121006, abcam, HK; dilution 1:500) and slug (sc-10436, Santa Cruz, USA, dilution 1:200), mouse anti-human monoclonal antibody E-cadherin (ab1416, abcam, HK; dilution 1:500), N-cadherin (ab98952, abcam, HK; dilution 1:500) and GADPH (ab8245, Abcam, HK; 1:1000) and mouse anti-human polyclonal antibody β-actin (ab20272, Abcam, HK; 1:500). After incubation with the secondary antibody labeled with HRP at room temperature for 2 h, protein bands were visualized using enhanced chemiluminescence (ECL) and detected using the BioImaging System.