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Jackson Laboratory mouse primary hippocampal and cortical neurons
$PLPPR3 and BASP1 localize to the presynaptic terminal (A) Phosphorylation of PLPPR3 S351 can be triggered in neurons. DIV8 primary <t>hippocampal</t> neurons were stimulated with Forskolin (30 μM, 5 min), and analyzed by western blot using indicated antibodies. (B) Quantification of the results in A ( n = 3). Data are represented as mean ± SEM. ∗∗∗∗ p < 0.0001, unpaired t test. (C) Localization of PLPPR3 and pS351 to synaptosomal fractions. Crude synaptosomes were prepared from adult mouse brain and analyzed by western blot. S2, cytosolic fraction; P2, crude synaptosomes; SYP1, Synaptophysin1. (D) Localization of PLPPR3 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. PLPPR3 was stained with our custom made anti-PLPPR3 antibody in combination with anti-VGLUT1. (E) Co-localization of Synaptophysin1 and PLPPR3 ICDm following overexpression in primary hippocampal neurons. Neurons were transfected with recombinant proteins at DIV1 and analyzed at DIV7. (F) Quantification of PLPPR3 ICDm clusters inside Synaptophysin1-positive synapses. Synaptophysin1 was co-expressed with PLPPR3 ICDm or PLPPR3 ICDm S351A. Graph shows PLPPR3 ICDm (WT or S351A) in Synaptophysin1-positive synapses. N = 3, n ≥ 7 neurons. Data are represented as mean ± SEM. (G) Localization of BASP1 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. BASP1 was stained with the custom made anti-BASP1 antibody in combination with anti-VGLUT1. (H) Co-localization of Synaptophysin1 and BASP1 in WT hippocampal neurons. (I) Co-localization of PLPPR3 ICDm and BASP1 in Plppr3−/− hippocampal neurons. In (H) and (I), neurons were transfected with recombinant proteins at DIV1 and imaged at DIV7. Scale bars, 5 μm. (J) Distance of BASP1 clusters to nearest VGLUT1 clusters (pre-synapses) in WT and PLPPR3 −/− neurons, and in PLPPR3 −/− neurons expression PLPPR3 ICDm or PLPPR3 ICDm S351A. Data are represented as mean ± SEM. (K–N) Examples of WT and PLPPR3 −/− neurons, and PLPPR3 −/− neurons expressing PLPPR3 ICDm or PLPPR3 ICDm S351A. Neurons were stained with anti-VGLUT1 and anti-BASP1 antibodies. Scale bars, 5 μm.$
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Article Title: Phosphorylation of presynaptic PLPPR3 controls synaptic vesicle release

Journal: iScience

doi: 10.1016/j.isci.2025.113435

$PLPPR3 and BASP1 localize to the presynaptic terminal (A) Phosphorylation of PLPPR3 S351 can be triggered in neurons. DIV8 primary hippocampal neurons were stimulated with Forskolin (30 μM, 5 min), and analyzed by western blot using indicated antibodies. (B) Quantification of the results in A ( n = 3). Data are represented as mean ± SEM. ∗∗∗∗ p < 0.0001, unpaired t test. (C) Localization of PLPPR3 and pS351 to synaptosomal fractions. Crude synaptosomes were prepared from adult mouse brain and analyzed by western blot. S2, cytosolic fraction; P2, crude synaptosomes; SYP1, Synaptophysin1. (D) Localization of PLPPR3 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. PLPPR3 was stained with our custom made anti-PLPPR3 antibody in combination with anti-VGLUT1. (E) Co-localization of Synaptophysin1 and PLPPR3 ICDm following overexpression in primary hippocampal neurons. Neurons were transfected with recombinant proteins at DIV1 and analyzed at DIV7. (F) Quantification of PLPPR3 ICDm clusters inside Synaptophysin1-positive synapses. Synaptophysin1 was co-expressed with PLPPR3 ICDm or PLPPR3 ICDm S351A. Graph shows PLPPR3 ICDm (WT or S351A) in Synaptophysin1-positive synapses. N = 3, n ≥ 7 neurons. Data are represented as mean ± SEM. (G) Localization of BASP1 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. BASP1 was stained with the custom made anti-BASP1 antibody in combination with anti-VGLUT1. (H) Co-localization of Synaptophysin1 and BASP1 in WT hippocampal neurons. (I) Co-localization of PLPPR3 ICDm and BASP1 in Plppr3−/− hippocampal neurons. In (H) and (I), neurons were transfected with recombinant proteins at DIV1 and imaged at DIV7. Scale bars, 5 μm. (J) Distance of BASP1 clusters to nearest VGLUT1 clusters (pre-synapses) in WT and PLPPR3 −/− neurons, and in PLPPR3 −/− neurons expression PLPPR3 ICDm or PLPPR3 ICDm S351A. Data are represented as mean ± SEM. (K–N) Examples of WT and PLPPR3 −/− neurons, and PLPPR3 −/− neurons expressing PLPPR3 ICDm or PLPPR3 ICDm S351A. Neurons were stained with anti-VGLUT1 and anti-BASP1 antibodies. Scale bars, 5 μm.$
Figure Legend Snippet: PLPPR3 and BASP1 localize to the presynaptic terminal (A) Phosphorylation of PLPPR3 S351 can be triggered in neurons. DIV8 primary hippocampal neurons were stimulated with Forskolin (30 μM, 5 min), and analyzed by western blot using indicated antibodies. (B) Quantification of the results in A ( n = 3). Data are represented as mean ± SEM. ∗∗∗∗ p < 0.0001, unpaired t test. (C) Localization of PLPPR3 and pS351 to synaptosomal fractions. Crude synaptosomes were prepared from adult mouse brain and analyzed by western blot. S2, cytosolic fraction; P2, crude synaptosomes; SYP1, Synaptophysin1. (D) Localization of PLPPR3 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. PLPPR3 was stained with our custom made anti-PLPPR3 antibody in combination with anti-VGLUT1. (E) Co-localization of Synaptophysin1 and PLPPR3 ICDm following overexpression in primary hippocampal neurons. Neurons were transfected with recombinant proteins at DIV1 and analyzed at DIV7. (F) Quantification of PLPPR3 ICDm clusters inside Synaptophysin1-positive synapses. Synaptophysin1 was co-expressed with PLPPR3 ICDm or PLPPR3 ICDm S351A. Graph shows PLPPR3 ICDm (WT or S351A) in Synaptophysin1-positive synapses. N = 3, n ≥ 7 neurons. Data are represented as mean ± SEM. (G) Localization of BASP1 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. BASP1 was stained with the custom made anti-BASP1 antibody in combination with anti-VGLUT1. (H) Co-localization of Synaptophysin1 and BASP1 in WT hippocampal neurons. (I) Co-localization of PLPPR3 ICDm and BASP1 in Plppr3−/− hippocampal neurons. In (H) and (I), neurons were transfected with recombinant proteins at DIV1 and imaged at DIV7. Scale bars, 5 μm. (J) Distance of BASP1 clusters to nearest VGLUT1 clusters (pre-synapses) in WT and PLPPR3 −/− neurons, and in PLPPR3 −/− neurons expression PLPPR3 ICDm or PLPPR3 ICDm S351A. Data are represented as mean ± SEM. (K–N) Examples of WT and PLPPR3 −/− neurons, and PLPPR3 −/− neurons expressing PLPPR3 ICDm or PLPPR3 ICDm S351A. Neurons were stained with anti-VGLUT1 and anti-BASP1 antibodies. Scale bars, 5 μm.

Techniques Used: Phospho-proteomics, Western Blot, Microscopy, Staining, Over Expression, Transfection, Recombinant, Expressing

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Journal: iScience

Article Title: Phosphorylation of presynaptic PLPPR3 controls synaptic vesicle release

doi: 10.1016/j.isci.2025.113435

Figure Lengend Snippet: PLPPR3 and BASP1 localize to the presynaptic terminal (A) Phosphorylation of PLPPR3 S351 can be triggered in neurons. DIV8 primary hippocampal neurons were stimulated with Forskolin (30 μM, 5 min), and analyzed by western blot using indicated antibodies. (B) Quantification of the results in A ( n = 3). Data are represented as mean ± SEM. ∗∗∗∗ p < 0.0001, unpaired t test. (C) Localization of PLPPR3 and pS351 to synaptosomal fractions. Crude synaptosomes were prepared from adult mouse brain and analyzed by western blot. S2, cytosolic fraction; P2, crude synaptosomes; SYP1, Synaptophysin1. (D) Localization of PLPPR3 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. PLPPR3 was stained with our custom made anti-PLPPR3 antibody in combination with anti-VGLUT1. (E) Co-localization of Synaptophysin1 and PLPPR3 ICDm following overexpression in primary hippocampal neurons. Neurons were transfected with recombinant proteins at DIV1 and analyzed at DIV7. (F) Quantification of PLPPR3 ICDm clusters inside Synaptophysin1-positive synapses. Synaptophysin1 was co-expressed with PLPPR3 ICDm or PLPPR3 ICDm S351A. Graph shows PLPPR3 ICDm (WT or S351A) in Synaptophysin1-positive synapses. N = 3, n ≥ 7 neurons. Data are represented as mean ± SEM. (G) Localization of BASP1 to presynaptic terminals in DIV16 WT primary hippocampal neurons analyzed by STED microscopy. BASP1 was stained with the custom made anti-BASP1 antibody in combination with anti-VGLUT1. (H) Co-localization of Synaptophysin1 and BASP1 in WT hippocampal neurons. (I) Co-localization of PLPPR3 ICDm and BASP1 in Plppr3−/− hippocampal neurons. In (H) and (I), neurons were transfected with recombinant proteins at DIV1 and imaged at DIV7. Scale bars, 5 μm. (J) Distance of BASP1 clusters to nearest VGLUT1 clusters (pre-synapses) in WT and PLPPR3 −/− neurons, and in PLPPR3 −/− neurons expression PLPPR3 ICDm or PLPPR3 ICDm S351A. Data are represented as mean ± SEM. (K–N) Examples of WT and PLPPR3 −/− neurons, and PLPPR3 −/− neurons expressing PLPPR3 ICDm or PLPPR3 ICDm S351A. Neurons were stained with anti-VGLUT1 and anti-BASP1 antibodies. Scale bars, 5 μm.

Article Snippet: Mouse: Primary hippocampal and cortical neurons (C57 Bl/6NCrl ) , The Jackson Laboratory , Cat#000664; RRID: IMSR_JAX:000664.

Techniques: Phospho-proteomics, Western Blot, Microscopy, Staining, Over Expression, Transfection, Recombinant, Expressing