golph3 polyclonal antibody  (Proteintech)

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Schematic of the CRISPR/Cas9-mediated strategy used to generate GOLPH3 knockout (GOLPH3 -/- ) mice by deleting exons 2 and 3. The diagnostic PCR (right) confirms wild-type (766 bp) and KO (382 bp) alleles in genomic DNA from mice of the indicated genotypes. B. Western blot analysis of liver lysates from male (top) and female (bottom) mice with the indicated genotypes. GOLPH3 and Tubulin (loading control) were detected with specific antibodies, confirming complete loss of GOLPH3 protein in knockout livers. C. Volcano plot showing differential protein abundance in livers from GOLPH3 +/+ vs GOLPH3 -/- mice (n = 3) by TMT-based quantitative proteomics. GOLPH3 is the most significantly downregulated protein. D. Volcano plot showing changes in O-glycopeptide abundance from the same samples as in (C), enriched using jacalin-based lectin weak affinity chromatography (LWAC) and analyzed by TMT-MS. Several known GALNT2 substrates (e.g., Kng1, Apoc4, Lamp1) show reduced glycosylation in GOLPH3 -/- livers. E. Diagram of the mucin-type O-glycosylation pathway, illustrating key enzymes and intermediates. GALNTs initiate glycosylation by transferring GalNAc to Ser/Thr residues. F. Schematic showing O-glycosylation sites in glycoproteins (e.g., Kng1, Apoc4, Lamp1) with decreased glycopeptide abundance in GOLPH3 -/- livers. Red arrowheads indicate a decrease; cyan equality symbols indicate no change. G. Volcano plot depicting differential lipid species abundance between GOLPH3 +/+ and GOLPH3 -/- livers (n = 10). Hexosylceramides (HexCer, blue) are elevated, while triglycerides (TAGs, red) are reduced in GOLPH3 -/- males. H. Box-and-whisker plots showing normalized abundance of major lipid classes. HexCer levels are significantly increased in the experimental group compared to controls (unpaired t-test, ***p < 0.001), while SM shows minor differences (n = 10 per group) I. Quantification of individual GlcCer and GalCer species via targeted LC-MS/MS across genotypes. GlcCer species are significantly elevated in GOLPH3 -/- livers; GalCer levels show minor increases (two-way ANOVA, *p < 0.05; **p < 0.001; ****p < 0.0001). J. Quantification of glycosphingolipid intermediates and products via LC-MS/MS. GlcCer accumulates in GOLPH3 -/- livers, with no significant change in downstream LacCer and gangliosides, consistent with impaired LCS activity (two-way ANOVA, * ****p < 0.0001). K. Schematic of the glycosphingolipid biosynthesis pathway. GOLPH3-dependent LCS converts GlcCer to LacCer, which serves as a precursor for downstream gangliosides and globosides. Accumulation of GlcCer in GOLPH3 -/- mice indicates reduced LCS function.

Article Snippet: The generation of GOLPH3 -/- mice was done by CRISPR/Cas-mediated genome engineering, generated in collaboration with Cyagen Biosciences, Santa Clara, CA, USA.

Techniques: CRISPR, Knock-Out, Diagnostic Assay, Western Blot, Control, Quantitative Proteomics, Glycoproteomics, Affinity Chromatography, Transferring, Whisker Assay, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Pedigree chart showing genotype frequencies among offspring from GOLPH3 +/- × GOLPH3 +/- crosses. B. Growth curves displaying body weight trajectories for male (left) and female (right) GOLPH3 +/+ , GOLPH3 +/- , and GOLPH3 -/- mice between 3 and 9 weeks of age. Data are presented as mean ± SEM. C. Representative whole-body micro-computed tomography (µCT) scans of 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice (left). Quantification of body length is shown on the right. Data are mean ± SEM (unpaired t-test, ***p < 0.001). Bar = 10mm. D. Representative µCT scans of the skull from 9-week-old male GOLPH3 +/+ and GOLPH3 -/- mice. Red arrows highlight craniofacial anomalies, including reduced snout length and diminished cranial bulging in GOLPH3 -/- mice. Bar = 5mm. E. Quantification of skull length-to-width ratio in 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Data are mean ± SEM (unpaired t-test, **p < 0.01). F. Representative hematoxylin and eosin (H&E)-stained coronal sections of the nasal cavity in 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Bar = 1mm. G. Quantification of skull bone density in GOLPH3 +/+ and GOLPH3 -/- mice assessed by µCT. Data are mean ± SEM (unpaired t-test, ***p < 0.001). H. Representative photograph of femurs and tibias from 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. I. Representative Alcian Blue-stained decalcified femur sections from 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Insets show magnified views of cortical bone regions. Bar = 500µm. J. µCT-based quantification of femoral cortical thickness in male (top) and female (bottom) GOLPH3 +/+ and GOLPH3 -/- mice. Data are mean ± SEM (unpaired t-test, ***p < 0.001; ****p<0.0001). K. Representative 3D reconstructions of femoral trabecular bone from 9-week-old male GOLPH3 +/+ and GOLPH3 -/- mice. Bar = 200µm L. Quantification of femoral trabecular bone volume fraction (%) in male (top) and female (bottom) mice. Data are mean ± SEM (unpaired t-test, ****p < 0.0001).

Article Snippet: The generation of GOLPH3 -/- mice was done by CRISPR/Cas-mediated genome engineering, generated in collaboration with Cyagen Biosciences, Santa Clara, CA, USA.

Techniques: Micro-CT, Staining

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Structure of GOLPH3 (left) and snapshots from coarse-grained MD simulations with membranes containing 5% (middle) or 10% (right) PtdIns(4)P. At 5%, GOLPH3 (purple surface) interacts via its β-hairpin (green), PtdIns(4) P -binding residues (W81, R90, R171, R174; orange), and Trp161 (blue). At 10% and higher, binding is mediated by a positively charged surface. Lipid head groups are shown as grey spheres; other lipids and water are omitted for clarity. B. Average number of contacts between GOLPH3 residues and membranes with 5% PtdIns(4)P across MD trajectories. C. MD-based contact analysis of GOLPH3 with membranes containing 0%, 10% (detected in 60% of replicates), 20%, 40% PtdIns(4) P , or 10% PtdIns(4,5) P ₂. D. S-acylation of endogenous GOLPH3 in HEK and HeLa cells. Palmitoylated proteins were detected by hydroxylamine treatment (+HA), followed by SDS-PAGE and anti-GOLPH3 immunoblotting. Calnexin was used as a loading control (Top). Stoichiometry of GOLPH3 palmitoylation determined via PEG-5 labeling and anti-GOLPH3 western blotting (Bottom). E. Palmitoylation analysis of GOLPH3 cysteine mutants. GOLPH3 KO HeLa cells were transfected with WT or mutant constructs, labeled with ³H-palmitic acid, and analyzed by autoradiography following immunoprecipitation. Quantification (mean ± SEM, n = 4) is relative to endogenous WT GOLPH3. F. Immunofluorescence of HeLa cells expressing WT or mutant GOLPH3, labeled with antibodies against GOLPH3, GM130 (Golgi), and Hoechst (nuclei). Scale bars = 10 μm (Top). Quantification of Golgi-to-cytosol GOLPH3 intensity ratio (Bottom). Whiskers show 2.5th–97.5th percentile; outliers as dots. One-way ANOVA vs. WT: ****p < 0.0001. Only transfected cells were analyzed (defined by GOLPH3 intensity > mean + 10 SD of non-transfected cells). G. MD model of S-acylated GOLPH3 (C84) interacting with membranes. H. Proposed model of GOLPH3 membrane recruitment, integrating electrostatic interactions (via PtdIns(4) P -binding residues or a positively charged surface), β-hairpin insertion, and S-acylation at C84, C108, and C122.

Article Snippet: The generation of GOLPH3 -/- mice was done by CRISPR/Cas-mediated genome engineering, generated in collaboration with Cyagen Biosciences, Santa Clara, CA, USA.

Techniques: Binding Assay, SDS Page, Western Blot, Control, Labeling, Transfection, Mutagenesis, Construct, Autoradiography, Immunoprecipitation, Immunofluorescence, Expressing, Membrane

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Overlay of 2D 1 H– 15 N HSQC spectra of 15 N-labeled GOLPH3 in its apo form (grey) and with increasing molar ratios of LCS peptide (1:1 to 4:1, gradient grey to violet). Assigned residues are labeled; boxes highlight regions with notable chemical shift changes. B. NMR-mapped LCS binding site on the negatively charged surface of GOLPH3, involving residues S246, D247, D258, E259, Q260, Y261, L263, T265, K266, F296, T297, and K298. C. Structural models of the GOLPH3–LCS complex from HADDOCK (guided by NMR data) and AlphaFold-Multimer (unguided). Insets show electrostatic interactions. LCS is in green; GOLPH3 residues with NMR shifts are in purple, others in grey. D. ITC binding curves for LCS WT with GOLPH3 D247R (left) and D247R/D258R/D262R (middle), and LCS R9A/R12A mutant with GOLPH3 WT (right). Representative of 2–3 replicates. E. Normalized contact heatmaps showing residue-level interactions between GOLPH3 and LCS during CG MD simulations. F. CG MD snapshot of S-acylated GOLPH3 (at C84) bound to the cytosolic tail and transmembrane domain of LCS (green) at the membrane.

Article Snippet: The generation of GOLPH3 -/- mice was done by CRISPR/Cas-mediated genome engineering, generated in collaboration with Cyagen Biosciences, Santa Clara, CA, USA.

Techniques: Labeling, Binding Assay, Mutagenesis, Residue, Membrane

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Quantitative immunofluorescence of LCS-SI-GFP Golgi retention in GOLPH3 KO HeLa cells transfected with various GOLPH3 variants. Scale bar: 10 µm. Staining: anti-GOLPH3 (cyan), anti-GFP before permeabilization (yellow), LCS-SI-GFP (magenta). B. Scatterplot showing total LCS-SI-GFP fluorescence (Total Cell intensity) and surface-exposed signal (PM intensity). Cells were classified as transfected or non-transfected based on GOLPH3 signal. Dashed lines indicate the intensity range used for downstream analysis. C. Quantification of LCS-SI-GFP Golgi retention in cells expressing different GOLPH3 mutants, as in (B).

Article Snippet: The generation of GOLPH3 -/- mice was done by CRISPR/Cas-mediated genome engineering, generated in collaboration with Cyagen Biosciences, Santa Clara, CA, USA.

Techniques: Immunofluorescence, Transfection, Staining, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet:

Article Snippet: The generation of GOLPH3 -/- mice was done by CRISPR/Cas-mediated genome engineering, generated in collaboration with Cyagen Biosciences, Santa Clara, CA, USA.

Techniques:

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Schematic of the CRISPR/Cas9-mediated strategy used to generate GOLPH3 knockout (GOLPH3 -/- ) mice by deleting exons 2 and 3. The diagnostic PCR (right) confirms wild-type (766 bp) and KO (382 bp) alleles in genomic DNA from mice of the indicated genotypes. B. Western blot analysis of liver lysates from male (top) and female (bottom) mice with the indicated genotypes. GOLPH3 and Tubulin (loading control) were detected with specific antibodies, confirming complete loss of GOLPH3 protein in knockout livers. C. Volcano plot showing differential protein abundance in livers from GOLPH3 +/+ vs GOLPH3 -/- mice (n = 3) by TMT-based quantitative proteomics. GOLPH3 is the most significantly downregulated protein. D. Volcano plot showing changes in O-glycopeptide abundance from the same samples as in (C), enriched using jacalin-based lectin weak affinity chromatography (LWAC) and analyzed by TMT-MS. Several known GALNT2 substrates (e.g., Kng1, Apoc4, Lamp1) show reduced glycosylation in GOLPH3 -/- livers. E. Diagram of the mucin-type O-glycosylation pathway, illustrating key enzymes and intermediates. GALNTs initiate glycosylation by transferring GalNAc to Ser/Thr residues. F. Schematic showing O-glycosylation sites in glycoproteins (e.g., Kng1, Apoc4, Lamp1) with decreased glycopeptide abundance in GOLPH3 -/- livers. Red arrowheads indicate a decrease; cyan equality symbols indicate no change. G. Volcano plot depicting differential lipid species abundance between GOLPH3 +/+ and GOLPH3 -/- livers (n = 10). Hexosylceramides (HexCer, blue) are elevated, while triglycerides (TAGs, red) are reduced in GOLPH3 -/- males. H. Box-and-whisker plots showing normalized abundance of major lipid classes. HexCer levels are significantly increased in the experimental group compared to controls (unpaired t-test, ***p < 0.001), while SM shows minor differences (n = 10 per group) I. Quantification of individual GlcCer and GalCer species via targeted LC-MS/MS across genotypes. GlcCer species are significantly elevated in GOLPH3 -/- livers; GalCer levels show minor increases (two-way ANOVA, *p < 0.05; **p < 0.001; ****p < 0.0001). J. Quantification of glycosphingolipid intermediates and products via LC-MS/MS. GlcCer accumulates in GOLPH3 -/- livers, with no significant change in downstream LacCer and gangliosides, consistent with impaired LCS activity (two-way ANOVA, * ****p < 0.0001). K. Schematic of the glycosphingolipid biosynthesis pathway. GOLPH3-dependent LCS converts GlcCer to LacCer, which serves as a precursor for downstream gangliosides and globosides. Accumulation of GlcCer in GOLPH3 -/- mice indicates reduced LCS function.

Article Snippet: The coding sequences for all GOLPH3 variants, except for the GOLPH3 WT and GOLPH3 D247A mutants, were optimized for Escherichia coli ( E. coli ) expression (Genscript).

Techniques: CRISPR, Knock-Out, Diagnostic Assay, Western Blot, Control, Quantitative Proteomics, Glycoproteomics, Affinity Chromatography, Transferring, Whisker Assay, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Pedigree chart showing genotype frequencies among offspring from GOLPH3 +/- × GOLPH3 +/- crosses. B. Growth curves displaying body weight trajectories for male (left) and female (right) GOLPH3 +/+ , GOLPH3 +/- , and GOLPH3 -/- mice between 3 and 9 weeks of age. Data are presented as mean ± SEM. C. Representative whole-body micro-computed tomography (µCT) scans of 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice (left). Quantification of body length is shown on the right. Data are mean ± SEM (unpaired t-test, ***p < 0.001). Bar = 10mm. D. Representative µCT scans of the skull from 9-week-old male GOLPH3 +/+ and GOLPH3 -/- mice. Red arrows highlight craniofacial anomalies, including reduced snout length and diminished cranial bulging in GOLPH3 -/- mice. Bar = 5mm. E. Quantification of skull length-to-width ratio in 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Data are mean ± SEM (unpaired t-test, **p < 0.01). F. Representative hematoxylin and eosin (H&E)-stained coronal sections of the nasal cavity in 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Bar = 1mm. G. Quantification of skull bone density in GOLPH3 +/+ and GOLPH3 -/- mice assessed by µCT. Data are mean ± SEM (unpaired t-test, ***p < 0.001). H. Representative photograph of femurs and tibias from 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. I. Representative Alcian Blue-stained decalcified femur sections from 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Insets show magnified views of cortical bone regions. Bar = 500µm. J. µCT-based quantification of femoral cortical thickness in male (top) and female (bottom) GOLPH3 +/+ and GOLPH3 -/- mice. Data are mean ± SEM (unpaired t-test, ***p < 0.001; ****p<0.0001). K. Representative 3D reconstructions of femoral trabecular bone from 9-week-old male GOLPH3 +/+ and GOLPH3 -/- mice. Bar = 200µm L. Quantification of femoral trabecular bone volume fraction (%) in male (top) and female (bottom) mice. Data are mean ± SEM (unpaired t-test, ****p < 0.0001).

Article Snippet: The coding sequences for all GOLPH3 variants, except for the GOLPH3 WT and GOLPH3 D247A mutants, were optimized for Escherichia coli ( E. coli ) expression (Genscript).

Techniques: Micro-CT, Staining

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Structure of GOLPH3 (left) and snapshots from coarse-grained MD simulations with membranes containing 5% (middle) or 10% (right) PtdIns(4)P. At 5%, GOLPH3 (purple surface) interacts via its β-hairpin (green), PtdIns(4) P -binding residues (W81, R90, R171, R174; orange), and Trp161 (blue). At 10% and higher, binding is mediated by a positively charged surface. Lipid head groups are shown as grey spheres; other lipids and water are omitted for clarity. B. Average number of contacts between GOLPH3 residues and membranes with 5% PtdIns(4)P across MD trajectories. C. MD-based contact analysis of GOLPH3 with membranes containing 0%, 10% (detected in 60% of replicates), 20%, 40% PtdIns(4) P , or 10% PtdIns(4,5) P ₂. D. S-acylation of endogenous GOLPH3 in HEK and HeLa cells. Palmitoylated proteins were detected by hydroxylamine treatment (+HA), followed by SDS-PAGE and anti-GOLPH3 immunoblotting. Calnexin was used as a loading control (Top). Stoichiometry of GOLPH3 palmitoylation determined via PEG-5 labeling and anti-GOLPH3 western blotting (Bottom). E. Palmitoylation analysis of GOLPH3 cysteine mutants. GOLPH3 KO HeLa cells were transfected with WT or mutant constructs, labeled with ³H-palmitic acid, and analyzed by autoradiography following immunoprecipitation. Quantification (mean ± SEM, n = 4) is relative to endogenous WT GOLPH3. F. Immunofluorescence of HeLa cells expressing WT or mutant GOLPH3, labeled with antibodies against GOLPH3, GM130 (Golgi), and Hoechst (nuclei). Scale bars = 10 μm (Top). Quantification of Golgi-to-cytosol GOLPH3 intensity ratio (Bottom). Whiskers show 2.5th–97.5th percentile; outliers as dots. One-way ANOVA vs. WT: ****p < 0.0001. Only transfected cells were analyzed (defined by GOLPH3 intensity > mean + 10 SD of non-transfected cells). G. MD model of S-acylated GOLPH3 (C84) interacting with membranes. H. Proposed model of GOLPH3 membrane recruitment, integrating electrostatic interactions (via PtdIns(4) P -binding residues or a positively charged surface), β-hairpin insertion, and S-acylation at C84, C108, and C122.

Article Snippet: The coding sequences for all GOLPH3 variants, except for the GOLPH3 WT and GOLPH3 D247A mutants, were optimized for Escherichia coli ( E. coli ) expression (Genscript).

Techniques: Binding Assay, SDS Page, Western Blot, Control, Labeling, Transfection, Mutagenesis, Construct, Autoradiography, Immunoprecipitation, Immunofluorescence, Expressing, Membrane

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Overlay of 2D 1 H– 15 N HSQC spectra of 15 N-labeled GOLPH3 in its apo form (grey) and with increasing molar ratios of LCS peptide (1:1 to 4:1, gradient grey to violet). Assigned residues are labeled; boxes highlight regions with notable chemical shift changes. B. NMR-mapped LCS binding site on the negatively charged surface of GOLPH3, involving residues S246, D247, D258, E259, Q260, Y261, L263, T265, K266, F296, T297, and K298. C. Structural models of the GOLPH3–LCS complex from HADDOCK (guided by NMR data) and AlphaFold-Multimer (unguided). Insets show electrostatic interactions. LCS is in green; GOLPH3 residues with NMR shifts are in purple, others in grey. D. ITC binding curves for LCS WT with GOLPH3 D247R (left) and D247R/D258R/D262R (middle), and LCS R9A/R12A mutant with GOLPH3 WT (right). Representative of 2–3 replicates. E. Normalized contact heatmaps showing residue-level interactions between GOLPH3 and LCS during CG MD simulations. F. CG MD snapshot of S-acylated GOLPH3 (at C84) bound to the cytosolic tail and transmembrane domain of LCS (green) at the membrane.

Article Snippet: The coding sequences for all GOLPH3 variants, except for the GOLPH3 WT and GOLPH3 D247A mutants, were optimized for Escherichia coli ( E. coli ) expression (Genscript).

Techniques: Labeling, Binding Assay, Mutagenesis, Residue, Membrane

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Quantitative immunofluorescence of LCS-SI-GFP Golgi retention in GOLPH3 KO HeLa cells transfected with various GOLPH3 variants. Scale bar: 10 µm. Staining: anti-GOLPH3 (cyan), anti-GFP before permeabilization (yellow), LCS-SI-GFP (magenta). B. Scatterplot showing total LCS-SI-GFP fluorescence (Total Cell intensity) and surface-exposed signal (PM intensity). Cells were classified as transfected or non-transfected based on GOLPH3 signal. Dashed lines indicate the intensity range used for downstream analysis. C. Quantification of LCS-SI-GFP Golgi retention in cells expressing different GOLPH3 mutants, as in (B).

Article Snippet: The coding sequences for all GOLPH3 variants, except for the GOLPH3 WT and GOLPH3 D247A mutants, were optimized for Escherichia coli ( E. coli ) expression (Genscript).

Techniques: Immunofluorescence, Transfection, Staining, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet:

Article Snippet: The coding sequences for all GOLPH3 variants, except for the GOLPH3 WT and GOLPH3 D247A mutants, were optimized for Escherichia coli ( E. coli ) expression (Genscript).

Techniques:

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Schematic of the CRISPR/Cas9-mediated strategy used to generate GOLPH3 knockout (GOLPH3 -/- ) mice by deleting exons 2 and 3. The diagnostic PCR (right) confirms wild-type (766 bp) and KO (382 bp) alleles in genomic DNA from mice of the indicated genotypes. B. Western blot analysis of liver lysates from male (top) and female (bottom) mice with the indicated genotypes. GOLPH3 and Tubulin (loading control) were detected with specific antibodies, confirming complete loss of GOLPH3 protein in knockout livers. C. Volcano plot showing differential protein abundance in livers from GOLPH3 +/+ vs GOLPH3 -/- mice (n = 3) by TMT-based quantitative proteomics. GOLPH3 is the most significantly downregulated protein. D. Volcano plot showing changes in O-glycopeptide abundance from the same samples as in (C), enriched using jacalin-based lectin weak affinity chromatography (LWAC) and analyzed by TMT-MS. Several known GALNT2 substrates (e.g., Kng1, Apoc4, Lamp1) show reduced glycosylation in GOLPH3 -/- livers. E. Diagram of the mucin-type O-glycosylation pathway, illustrating key enzymes and intermediates. GALNTs initiate glycosylation by transferring GalNAc to Ser/Thr residues. F. Schematic showing O-glycosylation sites in glycoproteins (e.g., Kng1, Apoc4, Lamp1) with decreased glycopeptide abundance in GOLPH3 -/- livers. Red arrowheads indicate a decrease; cyan equality symbols indicate no change. G. Volcano plot depicting differential lipid species abundance between GOLPH3 +/+ and GOLPH3 -/- livers (n = 10). Hexosylceramides (HexCer, blue) are elevated, while triglycerides (TAGs, red) are reduced in GOLPH3 -/- males. H. Box-and-whisker plots showing normalized abundance of major lipid classes. HexCer levels are significantly increased in the experimental group compared to controls (unpaired t-test, ***p < 0.001), while SM shows minor differences (n = 10 per group) I. Quantification of individual GlcCer and GalCer species via targeted LC-MS/MS across genotypes. GlcCer species are significantly elevated in GOLPH3 -/- livers; GalCer levels show minor increases (two-way ANOVA, *p < 0.05; **p < 0.001; ****p < 0.0001). J. Quantification of glycosphingolipid intermediates and products via LC-MS/MS. GlcCer accumulates in GOLPH3 -/- livers, with no significant change in downstream LacCer and gangliosides, consistent with impaired LCS activity (two-way ANOVA, * ****p < 0.0001). K. Schematic of the glycosphingolipid biosynthesis pathway. GOLPH3-dependent LCS converts GlcCer to LacCer, which serves as a precursor for downstream gangliosides and globosides. Accumulation of GlcCer in GOLPH3 -/- mice indicates reduced LCS function.

Article Snippet: These cells were transfected with GOLPH3 CRISPR plasmids (sc-412973, Santa Cruz Biotechnology) by using JetPrime transfection reagents (Polyplus) following the manufacturer’s instructions.

Techniques: CRISPR, Knock-Out, Diagnostic Assay, Western Blot, Control, Quantitative Proteomics, Glycoproteomics, Affinity Chromatography, Transferring, Whisker Assay, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Pedigree chart showing genotype frequencies among offspring from GOLPH3 +/- × GOLPH3 +/- crosses. B. Growth curves displaying body weight trajectories for male (left) and female (right) GOLPH3 +/+ , GOLPH3 +/- , and GOLPH3 -/- mice between 3 and 9 weeks of age. Data are presented as mean ± SEM. C. Representative whole-body micro-computed tomography (µCT) scans of 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice (left). Quantification of body length is shown on the right. Data are mean ± SEM (unpaired t-test, ***p < 0.001). Bar = 10mm. D. Representative µCT scans of the skull from 9-week-old male GOLPH3 +/+ and GOLPH3 -/- mice. Red arrows highlight craniofacial anomalies, including reduced snout length and diminished cranial bulging in GOLPH3 -/- mice. Bar = 5mm. E. Quantification of skull length-to-width ratio in 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Data are mean ± SEM (unpaired t-test, **p < 0.01). F. Representative hematoxylin and eosin (H&E)-stained coronal sections of the nasal cavity in 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Bar = 1mm. G. Quantification of skull bone density in GOLPH3 +/+ and GOLPH3 -/- mice assessed by µCT. Data are mean ± SEM (unpaired t-test, ***p < 0.001). H. Representative photograph of femurs and tibias from 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. I. Representative Alcian Blue-stained decalcified femur sections from 9-week-old GOLPH3 +/+ and GOLPH3 -/- mice. Insets show magnified views of cortical bone regions. Bar = 500µm. J. µCT-based quantification of femoral cortical thickness in male (top) and female (bottom) GOLPH3 +/+ and GOLPH3 -/- mice. Data are mean ± SEM (unpaired t-test, ***p < 0.001; ****p<0.0001). K. Representative 3D reconstructions of femoral trabecular bone from 9-week-old male GOLPH3 +/+ and GOLPH3 -/- mice. Bar = 200µm L. Quantification of femoral trabecular bone volume fraction (%) in male (top) and female (bottom) mice. Data are mean ± SEM (unpaired t-test, ****p < 0.0001).

Article Snippet: These cells were transfected with GOLPH3 CRISPR plasmids (sc-412973, Santa Cruz Biotechnology) by using JetPrime transfection reagents (Polyplus) following the manufacturer’s instructions.

Techniques: Micro-CT, Staining

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Structure of GOLPH3 (left) and snapshots from coarse-grained MD simulations with membranes containing 5% (middle) or 10% (right) PtdIns(4)P. At 5%, GOLPH3 (purple surface) interacts via its β-hairpin (green), PtdIns(4) P -binding residues (W81, R90, R171, R174; orange), and Trp161 (blue). At 10% and higher, binding is mediated by a positively charged surface. Lipid head groups are shown as grey spheres; other lipids and water are omitted for clarity. B. Average number of contacts between GOLPH3 residues and membranes with 5% PtdIns(4)P across MD trajectories. C. MD-based contact analysis of GOLPH3 with membranes containing 0%, 10% (detected in 60% of replicates), 20%, 40% PtdIns(4) P , or 10% PtdIns(4,5) P ₂. D. S-acylation of endogenous GOLPH3 in HEK and HeLa cells. Palmitoylated proteins were detected by hydroxylamine treatment (+HA), followed by SDS-PAGE and anti-GOLPH3 immunoblotting. Calnexin was used as a loading control (Top). Stoichiometry of GOLPH3 palmitoylation determined via PEG-5 labeling and anti-GOLPH3 western blotting (Bottom). E. Palmitoylation analysis of GOLPH3 cysteine mutants. GOLPH3 KO HeLa cells were transfected with WT or mutant constructs, labeled with ³H-palmitic acid, and analyzed by autoradiography following immunoprecipitation. Quantification (mean ± SEM, n = 4) is relative to endogenous WT GOLPH3. F. Immunofluorescence of HeLa cells expressing WT or mutant GOLPH3, labeled with antibodies against GOLPH3, GM130 (Golgi), and Hoechst (nuclei). Scale bars = 10 μm (Top). Quantification of Golgi-to-cytosol GOLPH3 intensity ratio (Bottom). Whiskers show 2.5th–97.5th percentile; outliers as dots. One-way ANOVA vs. WT: ****p < 0.0001. Only transfected cells were analyzed (defined by GOLPH3 intensity > mean + 10 SD of non-transfected cells). G. MD model of S-acylated GOLPH3 (C84) interacting with membranes. H. Proposed model of GOLPH3 membrane recruitment, integrating electrostatic interactions (via PtdIns(4) P -binding residues or a positively charged surface), β-hairpin insertion, and S-acylation at C84, C108, and C122.

Article Snippet: These cells were transfected with GOLPH3 CRISPR plasmids (sc-412973, Santa Cruz Biotechnology) by using JetPrime transfection reagents (Polyplus) following the manufacturer’s instructions.

Techniques: Binding Assay, SDS Page, Western Blot, Control, Labeling, Transfection, Mutagenesis, Construct, Autoradiography, Immunoprecipitation, Immunofluorescence, Expressing, Membrane

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Overlay of 2D 1 H– 15 N HSQC spectra of 15 N-labeled GOLPH3 in its apo form (grey) and with increasing molar ratios of LCS peptide (1:1 to 4:1, gradient grey to violet). Assigned residues are labeled; boxes highlight regions with notable chemical shift changes. B. NMR-mapped LCS binding site on the negatively charged surface of GOLPH3, involving residues S246, D247, D258, E259, Q260, Y261, L263, T265, K266, F296, T297, and K298. C. Structural models of the GOLPH3–LCS complex from HADDOCK (guided by NMR data) and AlphaFold-Multimer (unguided). Insets show electrostatic interactions. LCS is in green; GOLPH3 residues with NMR shifts are in purple, others in grey. D. ITC binding curves for LCS WT with GOLPH3 D247R (left) and D247R/D258R/D262R (middle), and LCS R9A/R12A mutant with GOLPH3 WT (right). Representative of 2–3 replicates. E. Normalized contact heatmaps showing residue-level interactions between GOLPH3 and LCS during CG MD simulations. F. CG MD snapshot of S-acylated GOLPH3 (at C84) bound to the cytosolic tail and transmembrane domain of LCS (green) at the membrane.

Article Snippet: These cells were transfected with GOLPH3 CRISPR plasmids (sc-412973, Santa Cruz Biotechnology) by using JetPrime transfection reagents (Polyplus) following the manufacturer’s instructions.

Techniques: Labeling, Binding Assay, Mutagenesis, Residue, Membrane

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet: A. Quantitative immunofluorescence of LCS-SI-GFP Golgi retention in GOLPH3 KO HeLa cells transfected with various GOLPH3 variants. Scale bar: 10 µm. Staining: anti-GOLPH3 (cyan), anti-GFP before permeabilization (yellow), LCS-SI-GFP (magenta). B. Scatterplot showing total LCS-SI-GFP fluorescence (Total Cell intensity) and surface-exposed signal (PM intensity). Cells were classified as transfected or non-transfected based on GOLPH3 signal. Dashed lines indicate the intensity range used for downstream analysis. C. Quantification of LCS-SI-GFP Golgi retention in cells expressing different GOLPH3 mutants, as in (B).

Article Snippet: These cells were transfected with GOLPH3 CRISPR plasmids (sc-412973, Santa Cruz Biotechnology) by using JetPrime transfection reagents (Polyplus) following the manufacturer’s instructions.

Techniques: Immunofluorescence, Transfection, Staining, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Molecular Regulation and Physiological Role of GOLPH3-mediated Golgi retention

doi: 10.1101/2025.06.26.661665

Figure Lengend Snippet:

Article Snippet: These cells were transfected with GOLPH3 CRISPR plasmids (sc-412973, Santa Cruz Biotechnology) by using JetPrime transfection reagents (Polyplus) following the manufacturer’s instructions.

Techniques: