Extended Data Figure 9: SZT2 is essential for mTORC1 inactivation under nutrient-deprived conditions in mice and MEFs.
a, PCR genotyping of Szt2+/+, Szt2+/− and Szt2−/− mice. The expected sizes of wild-type (WT) or Szt2 mutant (KO) alleles are indicated by arrows. b, RT–PCR analysis of mRNA of Szt2 in Szt2+/+ and Szt2−/− MEFs. Primer pair 1 (Szt2-1) detects a region downstream of the gene trap, and primer pair 2 (Szt2-2) detects a region upstream of the gene trap. c, The birth weight of neonates from the indicated genotypes are shown. Data represent mean ± s.d., n = 10–25, two-tailed unpaired t-test. NS, not significant. d, e, Neonates were fasted for 2 h (d) or 10 h (e). Total cell lysates prepared from the indicated organs were analysed by immunoblotting. f, g, Szt2+/+ and Szt2−/− MEFs (<4 passages) were deprived of amino acids for 1 h (f), or amino acid plus glucose for 2 h (g) and stimulated with amino acids (f), or amino acid plus glucose (g) for 10 min where indicated. Total cell lysates were analysed by immunoblotting. h, Szt2+/+ and Szt2−/− MEFs (<4 passages) were cultured in complete medium or starved with amino acids for 16 h and analysed as in f. i, Szt2+/+ and Szt2−/− MEFs (<4 passages) were deprived of amino acids for 1 h and stimulated with amino acids for 10 min where indicated. The localization of mTOR and LAMP2 was determined by immunostaining. j, Szt2+/+ and Szt2−/− MEFs (<4 passages) were cultured in complete medium or serum-starved for 24 h and analysed as in f. Data (f–j) are representative of two independent experiments.
