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Fig. 1. Conditional knockout of Miz1 POZ/BTB domain in keratinocytes. (A) K14cre mediates efficient recombination of the Miz1 lox allele in primary keratinocytes. For genotyping, PCR was performed on genomic DNA from keratinocytes that were isolated from back skin of 1-day-old mice without subsequent culture, using primers indicated in supplementary material Fig. S1. Note that in K14Cre⁺/Miz1^+/lox keratinocytes, the wild-type fragment using primers 1 and 5 is inefficiently amplified because of competition in the PCR by the shorter fragment. The wild-type and loxP allele fragment amplified with primers 1 and 5 (about 1400 bp) differed only in 70 bp and could not be distinguished. (B) Levels of Miz1 mRNA in keratinocytes K14Cre⁺/Miz1^lox/lox and control mice. Miz1 mRNA was almost undetectable in K14Cre⁺/Miz1^lox/lox keratinocytes using primers 3 and 4, which are located in the region coding for the POZ/BTB domain. By contrast, when primers 6 and 7, which are located outside the POZ/BTB domain coding region, were used, a signal was obtained as in control mice, indicating the presence of a truncated Miz1 transcript lacking the POZ/BTB coding region. (C) Western blot analysis, using the anti-Miz1 antibody 10E2, of proteins immunoprecipitated from keratinocyte lysates using the anti-Miz1 antibody H-190. A truncated Miz1 protein could be detected in K14Cre⁺/Miz1^lox/lox and K14Cre⁺/Miz1^+/lox keratinocytes (arrow). Arrowhead indicates Miz1.

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