Figure 6: Asparagine levels regulate mRNA translation.
From: Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor
(a) Immunoblot of HeLa lysates with (shASNS+Dox) and without (shASNS−Dox) a 48-h induction of ASNS shRNA expression and HeLa lysates 48 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA (shASNS). Lysates were immunoblotted for ASNS, phospho-4E-BP1 (S65), total 4E-BP1, phospho-S6K (T389), total S6K, phospho-S6 (S235/236), total S6 and tubulin. Green arrow indicates phosphorylated form of 4E-BP1; red arrow indicates unphosphorylated form. (b) Immunoblot showing levels of eIF4E and 4E-BP1 immunoprecipitated with anti-4E-BP1 antibody (left panels) and in the input lysate (right panels) from HeLa cells 48 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA (shASNS). (c) Relative rates of 35S-methionine incorporation into newly synthesized protein in HeLa cells 72 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA (shASNS). Error bars denote s.d. of the mean (n=3). P values were calculated by the Student’s t-test: **P<0.01; ***P<0.001.
