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. 2021 Feb 19;220(4):e202010048. doi: 10.1083/jcb.202010048

Figure 7.

Figure 7.

MAPK11/14 transmit multiple types of stress to regulate phosphorylation of SNX27 at Ser51 and endocytic trafficking. (A) Pull-down assay to assess phosphorylation of SNX27 under different stimulation conditions. HEK293T cells were transfected with GST-tagged SNX27 for 24 h and treated with multiple stimulations: EBSS (for 4 h), E+B (100 nM for 4 h), LPS (50 ng/ml for 1 h), CCCP (20 µM for 4 h), TG (1 µM for 4 h), IL-6 (10 ng/ml for 1h), EGF (15 ng/ml for 1h), or ATP (5 mM for 1 h). Complete medium (con) was used as a negative control. The cell lysate was used for pull-down with GST-affinity resin, followed by immunoblotting for GST-SNX27 and p-SNX27-Ser51S. Representative of three independent iterations. (B) Confocal imaging for GLUT1 (gray) and LAMP2 (red) immunofluorescence staining in the stable GFP-Parkin (green) HeLa cells. The cells were treated with CCCP (20 µM for 4 h) or not. Representative images are shown. Scale bars: 10 mm. Pearson’s correlation coefficients for GLUT1 and LAMP2. Results for individual cells are plotted, along with the mean and SD for each group (n = 34); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. (C) Confocal imaging for GLUT1 (gray) and phalloidin (red) immunofluorescence staining in HeLa cells stably expressing GFP-LC3 (green). The cells were treated with classical stimulations as indicated in A. Complete medium (con) was used as a negative control. Representative images are shown. Scale bars: 10 mm. Quantification of GLUT1 immunofluorescence on phalloidin-positive cell membrane. Results for individual cells are plotted, along with the mean and SD for each group (n = 30); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. (D) Pull-down assay to assess how LPS treatment alters phosphorylation of SNX27 Ser51 in the presence or absence of different MAPK pathway inhibitors. HEK293T cells were transfected with GST-tagged SNX27 for 24 h, pretreated with or without various types of 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), and the ERK1/2 inhibitor U0126 (25 µM for 2 h). The cells were then treated or not with 50 ng/ml LPS for 1 h. Control cells (vector) were transfected with empty vector. One tenth of the cell lysate was prepared as input, and the rest was used for pull-down with GST-affinity resin, followed by immunoblotting for GST-SNX27 and p-SNX27-Ser51. Representative of three independent iterations. (E) Confocal imaging for Venus-GLUT1 (green) and phalloidin (red) immunofluorescence staining of HeLa cells. Cells were transfected with Venus-GLUT1 for 24 h, treated 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), and the ERK1/2 inhibitor U0126 (25 µM for 2 h). Cells were then starved for 4 h (EBSS or E+B). Representative images are shown. Scale bars: 10 mm. Pearson’s correlation coefficients for Venus-GLUT1 and phalloidin. Results for individual cells are plotted, along with the mean and SD for each group (con, n = 33 cells; EBSS, n = 28; E+B, n = 29; SB202190+E+B, n = 36; SB203580+E+B, n = 40; JNK-IN-8+E+B, n = 41; U0126+E+B, n = 32); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01. HSD, honestly significant difference; ns, not significant.