Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) HEK-293T or SH-SY5Y cells expressing TAP-tagged WT VPS35 were subjected to TAP methodology and Western blot analysis. Inputs and TAP fractions were probed with anti-VPS35 antibody. TAP purifies VPS35 more efficiently in HEK-293T cells compared to SH-SY5Y cells. ( B ) HEK-293T cells expressing TAP-tagged WT VPS35 or empty vector (EV) were subjected to TAP methodology followed by SDS-PAGE and silver staining. Endogenous VPS35 is observed slightly below VPS35-TAP band at ∼100 kDa. ( C ) STRING diagram of interacting proteins identified by LC-MS/MS analysis of WT VPS35-TAP. ( D ) HEK-293T cells expressing TAP-tagged WT VPS35 or EV were subjected to TAP methodology with HEPES-based buffers followed by Western blot analysis. Input and VPS35-TAP fractions were probed with anti-VPS35 or anti-VPS26 antibodies to confirm recovery of the retromer and then subjected to LC-MS/MS. ( E ) STRING diagram of interacting proteins identified by LC-MS/MS analysis of WT VPS35-TAP. Outside of the core retromer subunits, no known interacting proteins of VPS35 were identified. ( F ) Proportional Venn diagram demonstrating proteins identified in WT VPS35 TAP experiments using Tris– vs. HEPES-based buffers. ( G ) STRING diagram demonstrating the 7 proteins commonly identified between the two TAP experiments.

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Plasmid Preparation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) HEK-293T cells expressing TAP-tagged WT VPS35 treated with increasing concentrations of DSP were subjected to affinity purification with either streptavidin or calmodulin resin followed by Western blot analysis under reducing or non-reducing conditions. Input and purified fractions were probed with anti-VPS35 or anti-actin antibodies. Only 0.3 mM DSP treatment preserved binding of TAP-tagged VPS35 to both streptavidin and calmodulin resins. (B) HEK-293T cells expressing TAP-tagged VPS35 treated with 0.3 mM DSP were subjected to TAP methodology followed by Western blot analysis under reducing or non-reducing conditions. Input and TAP fractions were probed with anti-VPS35 or anti-WASH1 antibodies. WT VPS35-TAP eluate was subjected to LC-MS/MS analysis. ( C ) Proportional Venn diagram showing the common interacting proteins of WT VPS35 identified in Tris-based alone vs HEPES-based with DSP TAP experiments. ( D ) Proportional Venn diagram demonstrating proteins identified in HEPES-based WT VPS35-TAP assays with or without DSP treatment. The addition of reversible cross-linking greatly increased the number of interacting proteins. ( E , F ) GO and KEGG pathway analysis using the functional annotation tool DAVID of VPS35-interacting proteins identified using DSP-treated TAP assays in HEK-293T cells overexpressing TAP-tagged VPS35-WT. Top 10 GO terms for each category and top 10 KEGG Pathway terms, based on Bonferroni-corrected p-value and fold enrichment, are displayed. Bubble plots generated using SRplot. ( G ) Functional enrichment analysis using the Cytoscape plug-in ClueGo with GO biological process terms of proteins found in DSP-treated WT VPS35 TAP assays. Pathways with a p-value of <0.05 and a Kappa score of 0.4 were mapped. Two-sided hypergeometric statistical analysis test with Bonferroni step-down p-value correction was used. Node size corresponds to p-value.

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Expressing, Affinity Purification, Western Blot, Purification, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Generated

Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) Soluble hemi-brain extracts from 3-4 month-old WT, heterozygous and homozygous KI mice were subjected to IP with anti-VPS35 antibody (or isotype-matched control anti-V5 IgG) followed by Western blot analysis. Input and IP fractions were probed with anti-VPS35 and anti-VPS26 antibodies, prior to subjecting VPS35 IP samples to LC-MS/MS analysis. ( B ) Correlation analysis of VPS35-interacting proteins from WT vs homozygous D620N VPS35 KI reveal a high degree of correlation (R = 0.995). ( C ) Proportional Venn diagram demonstrating the strong overlap between interacting proteins identified in WT and homozygous D620N VPS35 KI brains. ( D ) Table highlighting peptide intensities across conditions for core retromer subunits, and three interacting proteins depleted in D620N VPS35 KI brain. ( E ) Soluble striatal extracts from 3-4 month-old WT and KI mice were subjected to IP with anti-VPS35 antibody (or isotype-matched control anti-V5 IgG) followed by Western blot analysis. Input and IP fractions were probed with anti-VPS35 and anti-VPS26 antibodies, prior to subjecting VPS35 IP samples to LC-MS/MS analysis. ( F ) Table outlining peptide intensities across conditions for core retromer subunits in striatal tissue of D620N VPS35 KI mice. ( G ) Triton-soluble fractions from striatum of adult WT or D620N VPS35 KI mice were subjected to Western blot analysis to monitor steady-state levels of core retromer subunits (VPS35, VPS26, VPS29), Rab7 and TBC1D5. ( H ) Graphs indicate densitometric analysis of protein levels normalized to actin or β-tubulin and expressed as fold-change compared to WT mice (mean ± SEM, n = 3 mice/group). Data are not significant ( P >0.05) by unpaired, two-tailed Student’s t -test.

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) Soluble striatal extracts from 3-4 month-old WT and D620N VPS35 KI mice were subjected to IP with anti-VPS35 antibody (or isotype-matched control anti-V5 IgG) followed by Western blot analysis. Input and IP fractions were probed with anti-VPS35 and anti-TBC1D5 antibodies confirming a TBC1D5 binding deficit in D620N VPS35 KI brain. Arrows indicate the position of TBC1D5 in the IP VPS35 samples and two non-specific bands in the control IP V5 sample. ( B ) HEK-293T cells expressing V5-tagged VPS35 variants (WT or D620N) were subjected to IP with anti-V5 antibody followed by Western blot analysis. Inputs and IPs were probed with anti-TBC1D5 or anti-V5 antibodies. ( C ) Graph indicates densitometric analysis of TBC1D5 levels in V5 IP, normalized to endogenous levels of TBC1D5 in the input, and expressed as a percent of the WT VPS35 IP condition (mean ± SEM, n = 3-4 experiments). ( D ) Representative confocal immunofluorescent images of HEK-293T cells co-labeled for endogenous TBC1D5 (red) and V5-tagged VPS35 variants (green). ( E ) Graph indicates Pearson’s correlation coefficients for TBC1D5 with WT or D620N VPS35 (mean ± SEM, n = 50 cells/condition). Data were analyzed by unpaired, two-tailed Student’s t -test (* P <0.05).

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Control, Western Blot, Binding Assay, Expressing, Labeling, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Parathyroid Hormone-Related Protein Negatively Regulates Tumor Cell Dormancy Genes in a PTHR1/Cyclic AMP-Independent Manner

doi: 10.3389/fendo.2018.00241

Figure Lengend Snippet: Neither parathyroid hormone (PTH) nor parathyroid hormone-related protein (PTHrP) bind to/activate cyclic AMP (cAMP) in MCF7 cells. (A) cAMP production in MCF7 cells following 12 min stimulation with PTH(1–34) or PTHrP(1–141), or positive controls forskolin, prostaglandin E 2 (PGE 2 ), or salmon calcitonin (sCT). Graphs = mean + SE. n = 3 replicates from independent experiments. ** p < 0.01, *** p < 0.001 vs no treatment by one-way ANOVA with multiple comparisons. (B) cAMP response element (CRE)-luciferase signal following 4 h stimulation with PTH or positive controls forskolin, prostaglandin E 2 (PGE 2 ), or sCT. Graphs = mean + SE. n = 3 replicates from independent experiments. *** p < 0.001 vs no treatment by one-way ANOVA with multiple comparisons. (C) Confocal images of stable MCF7 and UMR106-01 cells cultured on poly- l -lysine-coated glass coverslips and serum starved for 1 h prior to the addition of tetramethylrhodamine-labeled PTH(1–34) (PTH -TMR , 100 nM) for 15 min at 37°C. Cells were fixed in 4% PFA and immunostained for the endogenous retromer subunit, vacuolar protein sorting 35 (VPS35). Scale bar, 10 µm. Representative of n = 3 independent experiments.

Article Snippet: Cells were then incubated with anti-VPS35 antibody (Santa Cruz Biotechnology Inc.) for 1 h at room temperature, and washed in 0.2% BSA-PBS and 1× PBS prior to incubation with Alexa Fluor 488 anti-mouse secondary antibody (Molecular Probes ® , Invitrogen), for 45 min at room temperature.

Techniques: Luciferase, Cell Culture, Labeling

Journal: Molecular Neurodegeneration

Article Title: The PKCι-β-arrestin2 axis disrupts SORLA retrograde trafficking, driving its degradation and amyloid pathology in Alzheimer’s disease

doi: 10.1186/s13024-025-00865-6

Figure Lengend Snippet: βARR2 directly interacts with SORLA and competes for the retromer interaction with SORLA. A Direct interaction of SORLA with βARR2 and VPS26 detected by in vitro GST pull-down assays. GST-fused βARR2, VPS26 and SMAD2 (negative control) were incubated with in vitro translated biotin-labeled SORLA-C. Top , Colorimetric detection of SORLA-C. Free probe, 1/50 of input. Bottom , Western blot with a GST-antibody showing GST-fusion proteins. B Quantification of the amount of SORLA-C in the pull-down complexes with indicated the GST-fusion proteins, normalized to the amount of each GST-fusion protein in the reaction. The data are presented as fold differences relative to the amount of SORLA-C bound to GST-VPS26, which was set to 1.0. *, p < 0.05; **, p < 0.01; ****, p < 0.0001, by one-way ANOVA Tukey’s multiple comparisons test. C βARR2 and SORLA form a complex in intact cells. HEK293 cells expressing βARR2-GFP alone or together with Myc-SORLA were subjected to co-IP assays using a Myc antibody. Representative blots are shown. D βARR2 interacts with SORLA through the FANSHY motif. HEK293 cells co-expressing βARR2-GFP or VPS26-FLAG with WT or mutant SORLA were subjected to co-IP assays using a Myc antibody. F-A, F 2172 → A mutation; Y-A, Y 2177 → A mutation. E & F Expression of βARR2 reduces the SORLA-VPS26 interaction. HEK293 cells co-expressing Myc-tagged SORLA and FLAG-tagged VPS26, together with GFP-tagged βARR2 or its empty vector, were subjected to co-IP assays. Representative blots (E) and quantification of the fold change of VPS26 in the Myc-SORLA-IP complex (F) are shown. **, p < 0.01 by paired t test. G & H The interaction between SORLA and the retromer complex is enhanced in the brain of βARR2 deficient mice compared to WT mice. Cortical lysates prepared from WT or Arrb2 −/− (KO) mice were subjected to co-IP assays using a SORLA antibody. Representative blots (G) and quantification of the fold change in VPS35 levels within the SORLA IP complex (H) are shown. Quantification was normalized to the level of VPS35 in the IP buffer-soluble input and to the amount of IP’ed SORLA. *, p < 0.05 by paired t test. I , L βARR2 and the retromer complex reciprocally interact with SORLA. Lysates prepared from control and AD postmortem PFC were subjected to co-IP assays using a mouse SORLA antibody (BD Biosciences). Representative plots (I) and quantification of the relative levels of VPS35 (J) and βARR2 (K) in the SORLA-IP complex were shown. The amount of VPS35 in the SORLA-IP complex inversely correlates with that of βARR2 (L). *, p < 0.05 by unpaired t test. All data are mean ± SEM

Article Snippet: The total soluble input and bound proteins were then eluted in SDS sample buffer and subjected to Western blot analysis using antibodies against SORLA (rabbit, Protein Tech), VPS35 (goat, Origene) and βARR2 ([ ], gift from Dr. Jeffrey Benovic at Thomas Jefferson University).

Techniques: In Vitro, Negative Control, Incubation, Labeling, Western Blot, Expressing, Co-Immunoprecipitation Assay, Mutagenesis, Plasmid Preparation, Control

Journal: Cell Reports

Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

doi: 10.1016/j.celrep.2019.10.096

Figure Lengend Snippet: Internalized NL2 Co-localizes and Interacts with SNX27 and Retromer (A and B) Confocal images of antibody feeding in hippocampal neurons co-expressing HA NL2 with either SNX27 GFP (A) or VPS35 GFP (B). Arrowheads show examples of co-localization. Scale bars, 25 μm (whole cell) and 5 μm (soma). (C–F) Western blots of co-immunoprecipitation from rat brain lysate via endogenous NL2 (C–E) showing interaction with endogenous SNX27 (C), VPS35 (D), and VPS26 (E) or via endogenous SNX27 (F) showing interaction with endogenous NL2 and VPS35. IP, immunoprecipitation. Numbers on the left indicate molecular weight in kDa. (G) Western blot of GST pull-down from rat brain lysate. See also Figure S1 .

Article Snippet: Membranes were blocked for 1h in milk (PBS, 0.1% Tween, 4% milk), and then incubated overnight at 4°C with shaking in primary antibodies diluted in milk (1:1000 Rabbit-anti-Neuroligin2 (Synaptic Systems 129-202), 1:100 Rabbit-anti-VPS35 (Atlas antibodies, HPA040802, G), 1:500 Rabbit-anti-VPS35 (Abcam, ab97545, D and F), 1:2000 Rabbit-anti-VPS26 (Abcam, ab181352), 1:100 Rabbit-anti-SNX27 (Atlas antibodies, HPA045816, C and G), 1:100 Mouse-anti-SNX27 (Abcam, ab77799, F), 1:200 Mouse-anti-HA-tag (supernatant, clone 12CA5, produced in house), 1:500 Mouse-anti-GST (Neuromab, N100/13) or 1:500 Rabbit-anti-GFP (SantaCruz, sc-8334)).

Techniques: Expressing, Western Blot, Immunoprecipitation, Molecular Weight

Journal: Cell Reports

Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

doi: 10.1016/j.celrep.2019.10.096

Figure Lengend Snippet:

Article Snippet: Membranes were blocked for 1h in milk (PBS, 0.1% Tween, 4% milk), and then incubated overnight at 4°C with shaking in primary antibodies diluted in milk (1:1000 Rabbit-anti-Neuroligin2 (Synaptic Systems 129-202), 1:100 Rabbit-anti-VPS35 (Atlas antibodies, HPA040802, G), 1:500 Rabbit-anti-VPS35 (Abcam, ab97545, D and F), 1:2000 Rabbit-anti-VPS26 (Abcam, ab181352), 1:100 Rabbit-anti-SNX27 (Atlas antibodies, HPA045816, C and G), 1:100 Mouse-anti-SNX27 (Abcam, ab77799, F), 1:200 Mouse-anti-HA-tag (supernatant, clone 12CA5, produced in house), 1:500 Mouse-anti-GST (Neuromab, N100/13) or 1:500 Rabbit-anti-GFP (SantaCruz, sc-8334)).

Techniques: Transduction, Produced, Purification, Recombinant, Western Blot, Clone Assay, Subcloning, Mutagenesis, Software