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mouse anti vps35  (StressMarq)


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    Structured Review

    StressMarq mouse anti vps35
    ( A ) HEK-293T or SH-SY5Y cells expressing TAP-tagged WT <t>VPS35</t> were subjected to TAP methodology and Western blot analysis. Inputs and TAP fractions were probed <t>with</t> <t>anti-VPS35</t> 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.
    Mouse Anti Vps35, supplied by StressMarq, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+vps35/VPS35+Antibody/bio_rxiv__64898__2026__04__09__717005-319-10-13
    Average 94 stars, based on 3 article reviews
    mouse anti vps35 - by Bioz Stars, 2026-10
    94/100 stars

    Images

    1) Product Images from "Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35"

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

    Journal: bioRxiv

    doi: 10.64898/2026.04.09.717005

    ( 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.
    Figure Legend 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.

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

    ( 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.
    Figure Legend 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.

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

    ( 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.
    Figure Legend 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.

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

    ( 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).
    Figure Legend 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).

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

    Related Articles

    Expressing:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Western Blot:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Plasmid Preparation:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    SDS Page:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Silver Staining:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Affinity Purification:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Purification:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Binding Assay:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Functional Assay:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Generated:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Control:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Two Tailed Test:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..

    Labeling:

    Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35
    Article Snippet: Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).Rab7a plasmids containing full-length human Rab7a with a N-terminal EGFP and 2xHA tag were obtained from Addgene (WT, #28047; T22N, #28048; Q67L, #28049).. 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).. Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light chain-specific (Jackson Immunoresearch).Secondary HRP-conjugated antibodies used for Western blotting were: goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch), mouse anti-goat IgG, light chain-specific (Jackson Immunoresearch) and mouse anti-rabbit IgG, light..



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    ( A ) HEK-293T or SH-SY5Y cells expressing TAP-tagged WT <t>VPS35</t> were subjected to TAP methodology and Western blot analysis. Inputs and TAP fractions were probed <t>with</t> <t>anti-VPS35</t> 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.
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    Image Search Results


    ( 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.

    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

    ( 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.

    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

    ( 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.

    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

    ( 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).

    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

    Fluorescence imaging showing fraction of CTR1 (green) localized with respective endocytic vesicles (red) while DAPI (blue) marks the nucleus at copper (50 µM) and CDDP (25µM & 50µM). The Scale bar represents 10 microns. (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

    Journal: bioRxiv

    Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

    doi: 10.1101/2025.11.03.686439

    Figure Lengend Snippet: Fluorescence imaging showing fraction of CTR1 (green) localized with respective endocytic vesicles (red) while DAPI (blue) marks the nucleus at copper (50 µM) and CDDP (25µM & 50µM). The Scale bar represents 10 microns. (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

    Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

    Techniques: Fluorescence, Imaging, Immunofluorescence, Marker, Whisker Assay

    (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in HEK293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

    Journal: bioRxiv

    Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

    doi: 10.1101/2025.11.03.686439

    Figure Lengend Snippet: (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in HEK293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

    Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

    Techniques: Immunofluorescence, Marker, Whisker Assay

    Figure 1. VPS35/Retromer is involved in MT1-MMP trafficking and stability. A, Fluorescent microscopy images showing the localization of MT1-MMP-GFP in control cells versus VPS35-knockdown WM793 cells. B, Percentage of cells with perinuclear localization of MT1-MMP-GFP in VPS35 shRNA WM793 cells. C, Fluorescent microscopy images showing the cell membrane localization of MT1-MMP-GFP in control cells versus VPS35-knockdown 1205 Lu cells. D, Biotin assay showing a decrease in membrane-localized MT1-MMP in VPS35 shRNA WM793 cells. E, VPS35 shRNAs reduce total MT1-MMP levels in WM793 cells. F, VPS35 shRNA reduces total MT1-MMP levels in 1205Lu cells. G, VPS35 shRNA reduces total MT1-MMP levels in SW480 and SW620 colon cells. H, SNX27 shRNA reduces total MT1-MMP levels in WM793 cells. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Journal: Science China. Life sciences

    Article Title: VPS35/Retromer-dependent MT1-MMP regulation confers melanoma metastasis.

    doi: 10.1007/s11427-024-2884-2

    Figure Lengend Snippet: Figure 1. VPS35/Retromer is involved in MT1-MMP trafficking and stability. A, Fluorescent microscopy images showing the localization of MT1-MMP-GFP in control cells versus VPS35-knockdown WM793 cells. B, Percentage of cells with perinuclear localization of MT1-MMP-GFP in VPS35 shRNA WM793 cells. C, Fluorescent microscopy images showing the cell membrane localization of MT1-MMP-GFP in control cells versus VPS35-knockdown 1205 Lu cells. D, Biotin assay showing a decrease in membrane-localized MT1-MMP in VPS35 shRNA WM793 cells. E, VPS35 shRNAs reduce total MT1-MMP levels in WM793 cells. F, VPS35 shRNA reduces total MT1-MMP levels in 1205Lu cells. G, VPS35 shRNA reduces total MT1-MMP levels in SW480 and SW620 colon cells. H, SNX27 shRNA reduces total MT1-MMP levels in WM793 cells. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Article Snippet: Mouse antibodies against VPS35 (Santa Cruz Biotechnology, Dallas, USA, IP: 2 μg for 500 μg of total protein lysate, 1:200 for PLA), STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), p-STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), GAPDH (Santa Cruz Biotechnology, 1:1,000 for IB), SNX27 (Abcam, Cambridge, UK, 1:1,000 for IB).

    Techniques: Microscopy, Control, Knockdown, shRNA, Membrane

    Figure 2. VPS35/Retromer affects MT1-MMP protein stability through the lysosome pathway. A, Western blotting analysis of MT1-MMP stability following 10 μg mL −1 CHX treatment in control and VPS35 shRNA WM793 cells. B, Quantitative analysis of MT1-MMP stability in WM793 control and VPS35 shRNA cells. C, Western blotting analysis of MT1-MMP stability following 10 μg mL −1 CHX treatment in control and VPS35 shRNA SW480 cells. D, Quantitative analysis of MT1-MMP stability in control and VPS35 shRNA SW480 cells. E, Western blotting analysis of MT1-MMP levels following 5 μmol L −1 MG-132 and 100 nmol L −1 BFA1 treatment in control and VPS35 shRNA WM793 cells. F, Western blotting analysis of MT1-MMP levels following 5 μmol L −1 MG-132 and 100 nmol L −1 BFA1 treatment in control and VPS35 shRNA SW480 cells.

    Journal: Science China. Life sciences

    Article Title: VPS35/Retromer-dependent MT1-MMP regulation confers melanoma metastasis.

    doi: 10.1007/s11427-024-2884-2

    Figure Lengend Snippet: Figure 2. VPS35/Retromer affects MT1-MMP protein stability through the lysosome pathway. A, Western blotting analysis of MT1-MMP stability following 10 μg mL −1 CHX treatment in control and VPS35 shRNA WM793 cells. B, Quantitative analysis of MT1-MMP stability in WM793 control and VPS35 shRNA cells. C, Western blotting analysis of MT1-MMP stability following 10 μg mL −1 CHX treatment in control and VPS35 shRNA SW480 cells. D, Quantitative analysis of MT1-MMP stability in control and VPS35 shRNA SW480 cells. E, Western blotting analysis of MT1-MMP levels following 5 μmol L −1 MG-132 and 100 nmol L −1 BFA1 treatment in control and VPS35 shRNA WM793 cells. F, Western blotting analysis of MT1-MMP levels following 5 μmol L −1 MG-132 and 100 nmol L −1 BFA1 treatment in control and VPS35 shRNA SW480 cells.

    Article Snippet: Mouse antibodies against VPS35 (Santa Cruz Biotechnology, Dallas, USA, IP: 2 μg for 500 μg of total protein lysate, 1:200 for PLA), STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), p-STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), GAPDH (Santa Cruz Biotechnology, 1:1,000 for IB), SNX27 (Abcam, Cambridge, UK, 1:1,000 for IB).

    Techniques: Western Blot, Control, shRNA

    Figure 3. Inhibition of VPS35/Retromer increases MT1-MMP colocalization with lysosomes. A, Co-IP analysis shows that VPS35 interacts with MT1-MMP. B, Colocalization analysis of MT1-MMP and VPS35, scale bar, 5 μm. C, PLA co-localization analysis between VPS35 and MT1-MMP, scale bar, 10 μm. D, Co-localization analysis of MT1-MMP and the lysotracker, scale bar, 5 μm. E, Colocalization analysis of MT1-MMP and the lysosomal marker LAMP-1 in control and VPS35 shRNA cells, scale bar, 5 μm. F, Statistical analysis of the colocalization rate between MT1-MMP and the lysosomal marker LAMP-1 in control and VPS35 shRNA cells. G, Colocalization analysis of MT1-MMP and the late endosome marker RAB7 in control and VPS35 shRNA cells, scale bar, 5 μm. H, Statistical analysis of the colocalization rate between MT1-MMP and RAB7 in control and VPS35 shRNA cells. Statistical analysis was performed by using Student’s t-test. ns, not significant; ***, P<0.001.

    Journal: Science China. Life sciences

    Article Title: VPS35/Retromer-dependent MT1-MMP regulation confers melanoma metastasis.

    doi: 10.1007/s11427-024-2884-2

    Figure Lengend Snippet: Figure 3. Inhibition of VPS35/Retromer increases MT1-MMP colocalization with lysosomes. A, Co-IP analysis shows that VPS35 interacts with MT1-MMP. B, Colocalization analysis of MT1-MMP and VPS35, scale bar, 5 μm. C, PLA co-localization analysis between VPS35 and MT1-MMP, scale bar, 10 μm. D, Co-localization analysis of MT1-MMP and the lysotracker, scale bar, 5 μm. E, Colocalization analysis of MT1-MMP and the lysosomal marker LAMP-1 in control and VPS35 shRNA cells, scale bar, 5 μm. F, Statistical analysis of the colocalization rate between MT1-MMP and the lysosomal marker LAMP-1 in control and VPS35 shRNA cells. G, Colocalization analysis of MT1-MMP and the late endosome marker RAB7 in control and VPS35 shRNA cells, scale bar, 5 μm. H, Statistical analysis of the colocalization rate between MT1-MMP and RAB7 in control and VPS35 shRNA cells. Statistical analysis was performed by using Student’s t-test. ns, not significant; ***, P<0.001.

    Article Snippet: Mouse antibodies against VPS35 (Santa Cruz Biotechnology, Dallas, USA, IP: 2 μg for 500 μg of total protein lysate, 1:200 for PLA), STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), p-STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), GAPDH (Santa Cruz Biotechnology, 1:1,000 for IB), SNX27 (Abcam, Cambridge, UK, 1:1,000 for IB).

    Techniques: Inhibition, Co-Immunoprecipitation Assay, Marker, Control, shRNA

    Figure 4. VPS35/Retromer regulates gene expression and inflammatory response. A, Effects of VPS35 and SNX27 shRNA on MT1-MMP mRNA levels in WM793 cells. B, Effects of VPS35 shRNA on MT1-MMP mRNA levels in 1205Lu cells. C, Western blotting analysis of VPS35 shRNA in three batches of cells. D, Volcano of differentially expressed genes in WM793 control and VPS35 shRNA cells. E, Heatmap analysis of gene expression in WM793 control and VPS35 shRNA cells. F, q-PCR analysis of differentially expressed genes in WM793 control and VPS35 shRNA cells. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Abbreviation: amyloid beta precursor protein binding family A member (APBA1); baculoviral IAP repeat-containing protein 3 (BIRC3); Golgi membrane protein 2 (CASC4); C-C motif chemokine ligand 2 (CCL2); coiled-coil domain containing 3 (CCDC3); C-X-C motif chemokine ligand 2 (CXCL2); C-X-C motif chemokine ligand 3 (CXCL3); C-X-C motif chemokine ligand 8 (CXCL8); follicular dendritic cell secreted protein (FDCSP); GATA binding protein 4 (GATA4); glycoprotein nmb (GPNMB); interleukin 1 alpha (IL-1A); interleukin 1 beta (IL-1B); interleukin 11 (IL-11); interleukin 32 (IL-32); microsomal glutathione S-transferase 3 (MGST3); MAGE family member B2 (MAGB2); NLR family pyrin domain containing 1 (NLRP1); NLR family pyrin domain containing 3 (NLRP3); phospholipase A2 group VII (PLA2G7); pappalysin 1 (PAPPA); TNFAIP3 interacting protein 3 (TNIP3); VAMP associated protein A (VAPA); Wnt family member 5A (WNT5A).

    Journal: Science China. Life sciences

    Article Title: VPS35/Retromer-dependent MT1-MMP regulation confers melanoma metastasis.

    doi: 10.1007/s11427-024-2884-2

    Figure Lengend Snippet: Figure 4. VPS35/Retromer regulates gene expression and inflammatory response. A, Effects of VPS35 and SNX27 shRNA on MT1-MMP mRNA levels in WM793 cells. B, Effects of VPS35 shRNA on MT1-MMP mRNA levels in 1205Lu cells. C, Western blotting analysis of VPS35 shRNA in three batches of cells. D, Volcano of differentially expressed genes in WM793 control and VPS35 shRNA cells. E, Heatmap analysis of gene expression in WM793 control and VPS35 shRNA cells. F, q-PCR analysis of differentially expressed genes in WM793 control and VPS35 shRNA cells. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Abbreviation: amyloid beta precursor protein binding family A member (APBA1); baculoviral IAP repeat-containing protein 3 (BIRC3); Golgi membrane protein 2 (CASC4); C-C motif chemokine ligand 2 (CCL2); coiled-coil domain containing 3 (CCDC3); C-X-C motif chemokine ligand 2 (CXCL2); C-X-C motif chemokine ligand 3 (CXCL3); C-X-C motif chemokine ligand 8 (CXCL8); follicular dendritic cell secreted protein (FDCSP); GATA binding protein 4 (GATA4); glycoprotein nmb (GPNMB); interleukin 1 alpha (IL-1A); interleukin 1 beta (IL-1B); interleukin 11 (IL-11); interleukin 32 (IL-32); microsomal glutathione S-transferase 3 (MGST3); MAGE family member B2 (MAGB2); NLR family pyrin domain containing 1 (NLRP1); NLR family pyrin domain containing 3 (NLRP3); phospholipase A2 group VII (PLA2G7); pappalysin 1 (PAPPA); TNFAIP3 interacting protein 3 (TNIP3); VAMP associated protein A (VAPA); Wnt family member 5A (WNT5A).

    Article Snippet: Mouse antibodies against VPS35 (Santa Cruz Biotechnology, Dallas, USA, IP: 2 μg for 500 μg of total protein lysate, 1:200 for PLA), STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), p-STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), GAPDH (Santa Cruz Biotechnology, 1:1,000 for IB), SNX27 (Abcam, Cambridge, UK, 1:1,000 for IB).

    Techniques: Gene Expression, shRNA, Western Blot, Control, Protein Binding, Membrane, Binding Assay

    Figure 5. VPS35/Retromer regulates MT1-MMP transcription through IL-6/STAT3. A, KEGG enrichment analysis of differentially expressed genes in VPS35 shRNA WM793 cells. B, Western blotting analysis of p-STAT3 (Tyr705) levels in WM793 control and VPS35 shRNA cells. C, Western blotting analysis of p-STAT3 (Tyr705) levels after treating with IL-6 in WM793 control and VPS35 shRNA cells. D, Western blotting analysis and quantification of STAT3 nuclear localization in control and VPS35 shRNA WM793 cells. E, q-PCR analysis of MT1-MMP mRNA levels in WM793 control and STAT3 shRNA cells. F, Western blotting analysis of MT1-MMP protein levels in WM793 control and STAT3 shRNA cells. G, q-PCR analysis of MT1-MMP mRNA levels following ectopic STAT3 expression in VPS35 shRNA cells. H, Western blotting analysis of MT1-MMP protein levels following ectopic STAT3 expression in VPS35 shRNA cells. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Journal: Science China. Life sciences

    Article Title: VPS35/Retromer-dependent MT1-MMP regulation confers melanoma metastasis.

    doi: 10.1007/s11427-024-2884-2

    Figure Lengend Snippet: Figure 5. VPS35/Retromer regulates MT1-MMP transcription through IL-6/STAT3. A, KEGG enrichment analysis of differentially expressed genes in VPS35 shRNA WM793 cells. B, Western blotting analysis of p-STAT3 (Tyr705) levels in WM793 control and VPS35 shRNA cells. C, Western blotting analysis of p-STAT3 (Tyr705) levels after treating with IL-6 in WM793 control and VPS35 shRNA cells. D, Western blotting analysis and quantification of STAT3 nuclear localization in control and VPS35 shRNA WM793 cells. E, q-PCR analysis of MT1-MMP mRNA levels in WM793 control and STAT3 shRNA cells. F, Western blotting analysis of MT1-MMP protein levels in WM793 control and STAT3 shRNA cells. G, q-PCR analysis of MT1-MMP mRNA levels following ectopic STAT3 expression in VPS35 shRNA cells. H, Western blotting analysis of MT1-MMP protein levels following ectopic STAT3 expression in VPS35 shRNA cells. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

    Article Snippet: Mouse antibodies against VPS35 (Santa Cruz Biotechnology, Dallas, USA, IP: 2 μg for 500 μg of total protein lysate, 1:200 for PLA), STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), p-STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), GAPDH (Santa Cruz Biotechnology, 1:1,000 for IB), SNX27 (Abcam, Cambridge, UK, 1:1,000 for IB).

    Techniques: shRNA, Western Blot, Control, Expressing

    Figure 6. VPS35/Retromer promotes melanoma metastasis through MT1-MMP. A, Melanoma lung metastasis model via mouse tail vein injection. B, Western blotting analysis of VPS35 and MT1-MMP in 1205Lu cells. C, In vivo bioluminescence imaging and quantification of bioluminescence (luminescence intensity in p/s, ×10 6) shows pulmonary metastasis of melanoma in mice. D, Overall survival curves of melanoma lung metastasis mouse model. E, Hematoxylin and eosin (H&E) staining showed that VPS35 shRNA in 1205Lu melanoma cells reduced metastasis in nude mice. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ****, P<0.0001.

    Journal: Science China. Life sciences

    Article Title: VPS35/Retromer-dependent MT1-MMP regulation confers melanoma metastasis.

    doi: 10.1007/s11427-024-2884-2

    Figure Lengend Snippet: Figure 6. VPS35/Retromer promotes melanoma metastasis through MT1-MMP. A, Melanoma lung metastasis model via mouse tail vein injection. B, Western blotting analysis of VPS35 and MT1-MMP in 1205Lu cells. C, In vivo bioluminescence imaging and quantification of bioluminescence (luminescence intensity in p/s, ×10 6) shows pulmonary metastasis of melanoma in mice. D, Overall survival curves of melanoma lung metastasis mouse model. E, Hematoxylin and eosin (H&E) staining showed that VPS35 shRNA in 1205Lu melanoma cells reduced metastasis in nude mice. Statistical analysis was performed by using Student’s t-test. *, P<0.05; **, P<0.01; ****, P<0.0001.

    Article Snippet: Mouse antibodies against VPS35 (Santa Cruz Biotechnology, Dallas, USA, IP: 2 μg for 500 μg of total protein lysate, 1:200 for PLA), STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), p-STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), GAPDH (Santa Cruz Biotechnology, 1:1,000 for IB), SNX27 (Abcam, Cambridge, UK, 1:1,000 for IB).

    Techniques: Injection, Western Blot, In Vivo, Imaging, Staining, shRNA

    Figure 7. Expression of VPS35 and MT1-MMP in melanoma tissues and survival analysis in melanoma patients. A, Representative images of VPS35 and MT1-MMP immunohistochemical staining across different clark grades. B, Comparative analysis of VPS35 expression levels in melanomas of different Clark grades. C, Overall survival curves based on VPS35 expression in melanoma patients. D, Comparative analysis of MT1-MMP expression levels in melanomas of different Clark grades. E, Correlation analysis of VPS35 and MT1-MMP expression levels in melanoma tissue. F, Correlation analysis of VPS35 and MT1-MMP expression levels in the TCGA database. G, Schematic summary of VPS35/Retromer in MT1-MMP regulation. MT1-MMP, as a membrane protein, is regulated in its intracellular transport and recycling by VPS35/Retromer. Inhibition of VPS35/ Retromer restricts MT1-MMP distribution on the membrane and increases its colocalization with lysosomes, promoting MT1-MMP degradation. Furthermore, inhibition of VPS35/Retromer suppresses the IL-6/STAT3 signaling pathway, reduces STAT3 nuclear translocation, and decreases MT1-MMP mRNA levels. In summary, VPS35/Retromer regulates MT1-MMP levels through a dual mechanism, thereby influencing tumor metastasis. Statistical analysis was performed by using Student’s t-test for B and D, Pearson correlation coefficient analysis for E and F. *, P<0.05; **, P<0.01; ***, P<0.001.

    Journal: Science China. Life sciences

    Article Title: VPS35/Retromer-dependent MT1-MMP regulation confers melanoma metastasis.

    doi: 10.1007/s11427-024-2884-2

    Figure Lengend Snippet: Figure 7. Expression of VPS35 and MT1-MMP in melanoma tissues and survival analysis in melanoma patients. A, Representative images of VPS35 and MT1-MMP immunohistochemical staining across different clark grades. B, Comparative analysis of VPS35 expression levels in melanomas of different Clark grades. C, Overall survival curves based on VPS35 expression in melanoma patients. D, Comparative analysis of MT1-MMP expression levels in melanomas of different Clark grades. E, Correlation analysis of VPS35 and MT1-MMP expression levels in melanoma tissue. F, Correlation analysis of VPS35 and MT1-MMP expression levels in the TCGA database. G, Schematic summary of VPS35/Retromer in MT1-MMP regulation. MT1-MMP, as a membrane protein, is regulated in its intracellular transport and recycling by VPS35/Retromer. Inhibition of VPS35/ Retromer restricts MT1-MMP distribution on the membrane and increases its colocalization with lysosomes, promoting MT1-MMP degradation. Furthermore, inhibition of VPS35/Retromer suppresses the IL-6/STAT3 signaling pathway, reduces STAT3 nuclear translocation, and decreases MT1-MMP mRNA levels. In summary, VPS35/Retromer regulates MT1-MMP levels through a dual mechanism, thereby influencing tumor metastasis. Statistical analysis was performed by using Student’s t-test for B and D, Pearson correlation coefficient analysis for E and F. *, P<0.05; **, P<0.01; ***, P<0.001.

    Article Snippet: Mouse antibodies against VPS35 (Santa Cruz Biotechnology, Dallas, USA, IP: 2 μg for 500 μg of total protein lysate, 1:200 for PLA), STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), p-STAT3 (Santa Cruz Biotechnology, 1:1,000 for IB), GAPDH (Santa Cruz Biotechnology, 1:1,000 for IB), SNX27 (Abcam, Cambridge, UK, 1:1,000 for IB).

    Techniques: Expressing, Immunohistochemical staining, Staining, Membrane, Inhibition, Translocation Assay