Skip to main content
. 2018 Feb 7;9:63. doi: 10.3389/fphar.2018.00063

FIGURE 4.

FIGURE 4

Effects of the proteasomal inhibitor, MG132, and Nedd4.2 gene silencing on SNAT2 stability, and Me-AIB uptake in HeLa cells. (A) HeLa cells were transiently transfected with 10 μg of a pcDNA6 vector containing SNAT2V5 and incubated in buffer containing AAs but lacking or supplemented with MG132 (10 μM) for 8 h. After this period cells were either lysed for analysis of SNAT2V5 by immunoblotting or used to assay Me-AIB uptake. (B) HeLa cells were incubated in the absence or presence of 1× physiological AA mix for 8 h and then subsequently maintained or re-exposed to a 1× AA mix 6 h in the absence or presence of 10 μM MG132 prior to assaying Me-AIB uptake. Bar values (A and B) are mean ± SEM from three to five separate experiments with experiment conducted in triplicate. Asterisks indicate a significant difference between indicated bars P ≤ 0.05. (C) HeLa cells stably expressing shRNA targeting Nedd4.2 or a control shRNA (Scr) were incubated for 8 h in the absence or presence of AAs or in EBSS containing 2 mM of the indicated AA. Lysates (40 μg) were then subject to SDS–PAGE and immunoblotting with an anti-V5 antibody or antibodies directed against Nedd4.2 or GAPDH, which was used as a gel loading control. (D) HeLa cells were transiently transfected with 7KA-SNAT2V5 mutant, 24 h post-transfection cells were treated as in (C), and lysates immunoblotted with an anti-V5 antibody or GAPDH antibody. The blot is representative of two similar experiments.