R 173

Recombinant KLF6 protein was in vitro translated using the T 7 Quick Coupled TnT System (Promega). DNA binding assays were performed using either 3 μg of MA-10 nuclear extracts or 3 μL of in vitro-translated protein as described previously (Martin & Tremblay 2005 (link)). The 32 P-labelled double-stranded oligonucleotides used as probe were (the KLF element is underlined): sense 5′-ACT AAC CCC ACC CTT GAC CT-3′ and antisense 5′-AGG TCA AGG GTG GGG TTA GT-3′. For the competition experiments, 2-or 5-fold molar excess of double-stranded oligonucleotides (wild-type or mutated as described above for the promoter mutagenesis) were added to the reaction. For supershift/blocking experiments, 3 or 5 μg of normal rabbit IgG or a commercially available anti-KLF6 antiserum (R-173, Santa Cruz Biotechnologies) were added to the binding reaction.

Adult CD-1 mice (~40 day old) were obtained on site and killed by CO 2 inhalation. The testes were harvested and fixed with ice-cold 4% paraformaldehyde (w/v) for 24 h. Tissues were then dehydrated with ethanol, substituted with xylene, embedded in paraffin and cut into 5 µM sections. Following paraffin removal, tissues were blocked with 0.5% goat serum in PBS for 1 h at 25°C. Immunodetection was performed using an avidin-biotin approach according to the manufacturer's instructions for the Vectastain Elite ABC reagent (Vector Laboratories, Ontario, Canada). KLF6 protein localization was assessed using an anti-KLF6 rabbit polyclonal antiserum (R-173, 4 μg/mL; Santa Cruz Biotechnologies). Negative control corresponds to the same procedure except that the anti-KLF6 antibody was replaced with normal rabbit IgG (SC2027, 4 μg/mL, Santa Cruz Biotechnologies). Final detection was done using AEC (3-amino-9-ethylcarbazole from Sigma-Aldrich) as substrate and the sections were counterstained with hematoxylin Gill no.1 (VWR International, Mount-Royal, Québec, Canada). All experiments were conducted according to the Canadian Council for Animal Care and have been approved by the Animal Care and Ethics Committee of Laval University (protocol # 2009011).