Figure 7.
PI(3)P regulates the cell surface levels of SNX17- and SNX27-dependent cargoes. (A) Representative confocal images of DIV16 rat hippocampal neurons transfected at DIV15 with HA-VPS34. 24 h later, neurons were incubated live with antibodies against surface β1-integrin and surface GluA1 for 15 min at 37°C. Cells were then washed and further incubated at 37°C for 10 min. Antibodies remaining on the neuronal surface were stripped with an acid wash (0.5 M NaCl + 0.2 N acetic acid, 1 min, 4°C), followed by fixation, blocking, and incubation with secondary antibodies. Neurons were then permeabilized, blocked, and stained with an anti-HA antibody. Scale bar, 2.5 µm. (B) The percentage of GluA1 overlapping with β1-integrin was analyzed using Mander’s colocalization coefficient (×100). 61.90 ± 1.999%, N = 30 neurons. Three independent experiments. Error bar is SEM. (C) The percentage of β1-integrin overlapping with GluA1 was analyzed using Mander’s colocalization coefficient (×100). 66.80 ± 2.514%, N = 30 neurons. Three independent experiments. Error bar is SEM. (D) The percentage of endosomes containing both β1-integrin and GluA1 that colocalize with HA-VPS34 was analyzed using Mander’s colocalization coefficient (×100). 79.48 ± 1.283%, N = 30 neurons. Three independent experiments. Error bar is SEM. (E) DIV11 rat cortical neurons were infected with lentiviruses carrying ctrl- or VPS34-shRNAs, and the surface levels of β1-integrin and GluA1 were determined at DIV17 using surface biotinylation assays. The total levels of β1-integrin, GluA1, VPS34, SNX17, and SNX27 were validated by western blot of the lysate, and GAPDH was used as a loading control. (F) The levels of surface β1-integrin protein were quantified and expressed as a percentage of ctrl-shRNA. ctrl-shRNA: 100%, VPS34-shRNA: 57.40 ± 6.130%. N = 4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, ***P < 0.005. Error bars are SEM. (G) The levels of surface GluA1 protein were quantified and expressed as a percentage of ctrl-shRNA. ctrl-shRNA: 100%, VPS34-shRNA: 52.180 ± 7.024%. N = 3 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. DIV, days in vitro. Source data are available for this figure: SourceData F7.

PI(3)P regulates the cell surface levels of SNX17- and SNX27-dependent cargoes. (A) Representative confocal images of DIV16 rat hippocampal neurons transfected at DIV15 with HA-VPS34. 24 h later, neurons were incubated live with antibodies against surface β1-integrin and surface GluA1 for 15 min at 37°C. Cells were then washed and further incubated at 37°C for 10 min. Antibodies remaining on the neuronal surface were stripped with an acid wash (0.5 M NaCl + 0.2 N acetic acid, 1 min, 4°C), followed by fixation, blocking, and incubation with secondary antibodies. Neurons were then permeabilized, blocked, and stained with an anti-HA antibody. Scale bar, 2.5 µm. (B) The percentage of GluA1 overlapping with β1-integrin was analyzed using Mander’s colocalization coefficient (×100). 61.90 ± 1.999%, N = 30 neurons. Three independent experiments. Error bar is SEM. (C) The percentage of β1-integrin overlapping with GluA1 was analyzed using Mander’s colocalization coefficient (×100). 66.80 ± 2.514%, N = 30 neurons. Three independent experiments. Error bar is SEM. (D) The percentage of endosomes containing both β1-integrin and GluA1 that colocalize with HA-VPS34 was analyzed using Mander’s colocalization coefficient (×100). 79.48 ± 1.283%, N = 30 neurons. Three independent experiments. Error bar is SEM. (E) DIV11 rat cortical neurons were infected with lentiviruses carrying ctrl- or VPS34-shRNAs, and the surface levels of β1-integrin and GluA1 were determined at DIV17 using surface biotinylation assays. The total levels of β1-integrin, GluA1, VPS34, SNX17, and SNX27 were validated by western blot of the lysate, and GAPDH was used as a loading control. (F) The levels of surface β1-integrin protein were quantified and expressed as a percentage of ctrl-shRNA. ctrl-shRNA: 100%, VPS34-shRNA: 57.40 ± 6.130%. N = 4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, ***P < 0.005. Error bars are SEM. (G) The levels of surface GluA1 protein were quantified and expressed as a percentage of ctrl-shRNA. ctrl-shRNA: 100%, VPS34-shRNA: 52.180 ± 7.024%. N = 3 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t test, **P < 0.01. Error bars are SEM. DIV, days in vitro. Source data are available for this figure: SourceData F7.

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