Figure S5.

Multiple types of stress promote SNX27 phosphorylation via activating MAPK11/14. (A) Pull-down assay to assess Ser51 phosphorylation of SNX27 under erastin treatment. HEK293T cells were transfected with GST-tagged SNX27 for 24 h and treated with erastin (5 µM) for 6 h. Complete medium (con) was used as a negative control. The cell lysate was used for pull-down with GSH affinity resin, followed by immunoblotting for GST-SNX27 and p-SNX27-Ser51S. Representative of three independent iterations. (B) Pull-down assay to assess how starvation alters phosphorylation of SNX27 Ser51 in the presence or absence of different MAPK pathway inhibitors. HEK293T cells were transfected with GST-tagged SNX27 or empty vector (vector) for 24 h, pretreated with or without different pathway inhibitors: the MAPK11/14-specic inhibitor SB202190 (10 µM for 2 h), the p38 pathway inhibitor SB203580 (10 µM for 2 h), the JNK inhibitor JNK-IN-8 (5 µM for 2 h), the JNK inhibitor SP600125 (10 µM for 2 h), and the ERK1/2 inhibitor U0126 (25 µM for 2 h). Cells were then starved for 4 h. One tenth of the cell lysate was prepared as input, and the rest was used for pull-down with GSH affinity resin, followed by immunoblotting for GST-SNX27 and p-SNX27-Ser51. Representative of two independent iterations.