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. Author manuscript; available in PMC: 2017 Dec 7.
Published in final edited form as: Cell Rep. 2017 Dec 5;21(10):2895–2910. doi: 10.1016/j.celrep.2017.11.027

Fig. 6. GADD34 promotes SNAT2 maturation independently from the eIF2α phosphorylation status in mouse embryonic fibroblasts.

Fig. 6

Experiments were conducted in mouse embryonic fibroblasts expressing mutated eIF2α protein (S51A MEFs). A. Immunodetection of SNAT2 protein in extracts from cells exposed to 500 mOsm media for the indicated times. B. Immunodetection of SNAT2 in membrane fractions from cells treated as in panel A. C. 14C-MeAIB uptake in cells exposed to 500 mOsm media for 5h with or without Sal003 (30 μM) Sephin 1 (Seph, 100 μM) or Guanabenz (Guan, 100 μM). D. RT-qPCR analysis of SNAT2 mRNA levels from cells treated with 500 mOsm media for 5h in the presence of GADD34/PP1 inhibitors. Values were normalized to GAPDH mRNA and are plotted as a fold of induction over control. E. Western blot analysis of extracts from cells treated with 500 mOsm media for 5h in the presence of Sal003 (30 μM), Tunicamycin (Tu, 500 nM) or Golgicide A (GCA, 20 μM). A nonspecific band is indicated (#). Positions of protein size markers are marked. F. Western blot analysis of extracts from cells expressing shRNA against GADD34 and treated with 500 mOsm media. G. Quantification of Golgi fragmentation in cells incubated in the indicated stress conditions for 3h or treated with GADD34/PP1 inhibitors Sal003 (30 μM) or Sephin 1 (Seph, 50 μM) in control media for 2h. H. Immunofluorescent staining of F-actin (green channel) and α-tubulin (red channel) in control cell and after exposure to 500 mOsm media with or without Sephin 1 (Seph, 50 μM). Upper panel Scale bar is 20 μm. 3 lower panels represent magnification of boxed areas from the upper panel, with scale bar 5 μm. I. Immunodetection of SNAT2 protein in extracts from cells incubated in 500 mOsm for 5h with the indicated concentrations of nocodazole during the last 2h of treatment. J. Immunofluorescent staining of α-tubulin (red channel) and the Golgi marker-GM130 (green channel) in cells treated with 500 mOsm stress with or without nocodazole (Noc, 2 μM). Scale bar is 20 μm. Data in panels C and G are represented as mean of 3 experiments ± SD