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

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© 2021 Mao et al.

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Figure 3. — Starvation promotes SNX27 phosphorylation at Ser51. (A) Expression of SNX27 was determined by immunoblotting in the indicated HEK293T or HeLa cells stably expressing GFP-LC3. The cells were starved for 4 h in the presence or absence of cycloheximide (CHX; 50 µg/ml for 9 h). Representative images from one of three independent experiments are shown. The numbers under the bands represent the relative expression of SNX27 to β-actin. Band intensities were quantified with ImageJ and normalized to their respective control group. (B) Starvation increases phosphorylation of endogenous SNX27, determined by the pan-phospho-Ser/Thr antibody. HEK293T cells were starved for 4 h (E+B) or not. One tenth of the cell lysate was prepared as input, and the rest was used for immunoprecipitation with anti-SNX27 antibody, followed by immunoblotting using antibody against pan-phospho-Ser/Thr. IgG served as a negative control. Representative images from one of three independent experiments are shown. The numbers under the bands represent the relative ratio of pan-phospho-Ser/Thr to SNX27. Band intensities were quantified with ImageJ and normalized to the control group. (C) Full MS/MS spectrum for the identification of Ser51 phosphorylation on SNX27 (b and y ions indicate peptide backbone fragment ions). HEK293T cells stably expressing GST-SNX27 were starved (EBSS or E+B). GST-SNX27 was captured by GST-affinity resin, proteolyzed, and used for LC-MS/MS analysis. Changes in phosphorylation status were determined by label-free quantification, and the relative phosphorylation was normalized to total protein amount. MS/MS spectrum shows the phosphorylation at Ser51, and column chart represents the relative phosphorylation of SNX27 on Ser51, determined from two (EBSS) or three (E+B) independent experiments. Data are presented as means ± SD, and one-way ANOVA by Tukey’s HSD test or t test was used for data analysis. ***, P < 0.001. (D) Starvation increases phosphorylation of endogenous SNX27 on Ser51, determined by a phospho-Ser51-specific antibody. HEK293T cells were transiently transfected with GST-tagged SNX27 for 24 h. Cells expressing SNX27 proteins were starved or not. The cell lysate was used for pull-down with GST-affinity resin, followed by immunoblotting using antibodies against GST-SNX27 and p-SNX27-Ser51. Representative images from one of three independent experiments are shown. The numbers under the bands represent the relative level of phospho-SNX27 to GST-SNX27. Band intensities were quantified with ImageJ and normalized to control group. HSD, honestly significant difference; S51, Ser51.