Wild-type and knockout mice were created using embryonic stem cells from the 129P2 strain carrying a gene trap vector and injected into C57BL/6 blastocysts, as previously described (Calcraft et al. 2009 (link)). We confirmed that Tpcn2 is not transcribed in the knockout mice by running qPCR on multiple tissues in knockout and wild-type mice (see Figure S1). PCR was conducted as described above, using primers for GAPDH (forward 3′-CTGAAGGGCATCTTGGGCTA-5′ and reverse 3′-GCCGTATTCATTGTCATACCA-5′) and Tpcn2 (forward 3′-CCAGGCTACCTGTCCTACCA-5′ and reverse 3′-CAGGAAGCGAAACACAATCA-5′). Heterozygous mice were bred and set up as breeder pairs. Male Tpcn2 knockout, heterozygote, and wild-type mice from the heterozygote breeders were phenotyped at 9–13 weeks of age (n = 5–7 in each group), using the IPGTT, described above and in Solberg Woods et al. (2010 (link), 2012) (link). Blood was collected after an overnight fast and at 15, 30, 60, 90, and 120 min after a 1-g/kg glucose injection. We used the Ascensia Elite system for reading blood glucose values (Bayer, Elkhart, IN). We also collected blood at each time point for subsequent analysis of plasma insulin levels, which was assayed using an ultrasensitive ELISA kit from Alpco Diagnostics (Salem, NH).