Kallikrein 1b26 (klk1b26) protein and its mRNA levels in submandibular glands (SMGs) of male and female mice. (A) Western blot analysis of klk1b26 proteins in SMGs. One sample of SMG protein extract was prepared from one mouse (two SMGs). The protein extracts from male and female SMGs (10 ng protein each) were subjected to SDS-PAGE (16% non-reducing gel), and blotted onto a polyvinylidene difluoride (PVDF) filter. The klk1b26 protein was detected with anti-klk1b26 polyclonal antibody. Quantitative determination of the klk1b26 signals was made by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/). The klk1b26 protein ratio of male SMG to female SMG was estimated to be 9.16 ± 1.64 (mean ± SD; n = 3 mice). (B) Quantitative reverse transcription (RT)-PCR analysis of klk1b26 mRNA in SMGs. First-strand DNAs were prepared with 200 ng of total RNAs from male and female SMGs by using oligo(dT) primer. PCR was performed with primer pairs (F169/R552) targeting the middle region (169th to 552nd) of klk1b26 mRNA. The PCR conditions used were as follows: 94°C for 30 s, 55°C for 30 s, and 68°C for 120 s for denaturation, annealing, and elongation, respectively. (C) Quantitative determination of density of klk1b26 PCR products was made by computer-assisted image analysis, NIH image. The klk1b26 mRNA level of male SMG was estimated to be sevenfold higher than that of female SMG from the results of duplicate experiments.