Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Cell homogenates ( H ) from the indicated cell lines were used to prepare cytosolic ( C ) and membrane ( M ) fractions. Equivalent amounts of each fraction (10 μg of proteins) were subjected to SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (B) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in the cell homogenates as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in cytosolic ( C ) and membrane ( M ) fractions as shown in (A). Bar represents the mean ± standard deviation of the amount of immunoblot signal normalized with the signal for β-actin, and also for the total amount of protein in each fraction (for more details see ). *** P < 0.001; ns , not statistically significant.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) Samples of a membrane fraction (70 μg of proteins) from MCF 10A (A), MCF7 (B), and MDA-MB-231 (C) cells were incubated on ice for 1 hour with either 10 mM Tris HCl pH 7.4 ( Control ), 1 M KCl in 10 mM Tris HCl pH 7.4 ( KCl ) or 0.2 M Na 2 CO 3 pH 11.3 ( Na 2 CO 3 ). After centrifugation, pelleted membranes ( P ) and extracted proteins in the supernatant ( S ) were processed by SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. Syn16 , Syntaxin 16. The position of molecular mass markers is indicated on the left. (D-F) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in pellets ( P ) and supernatants ( S ) as shown in A-C of membranes incubated in control conditions (D), in 1 M KCl (E), or in 0.2 M Na 2 CO 3 (F). Bar represents the mean ± standard deviation of the amount of immunoblot signal. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Incubation, Control, Centrifugation, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: NRK (A and B), MCF 10A (C and D), MDA-MB-231 (E and F), and MCF7 (G and H) cells were left untreated ( Control ) or treated with 5 μg/ml BFA for 60 min ( BFA ). Cells were fixed, permeabilized, and immunolabeled with rabbit polyclonal antibody to GOLPH3, mouse monoclonal antibody to GM130, and either sheep antibody to TGN38 (A and B) or sheep antibody to TGN46 (C to H). Secondary antibodies were Alexa-594-conjugated donkey anti-rabbit IgG (red channel), Alexa-488-conjugated donkey anti-mouse IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Stained cells were examined by fluorescence microscopy. Merging red, green, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Insets show 1.7x magnifications. Bar, 10 μm.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Control, Immunolabeling, Staining, Fluorescence, Microscopy, Generated

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C and examined by fluorescence microscopy. The time after initiation of imaging is shown in the bottom right corner of each panel in minutes:seconds. Images are representative of 15–20 videos of up to 200 seconds of recording. In B, filled arrows indicate a vesicular structure moving from the Golgi to the periphery of the cell. In C, filled arrows indicate a vesicular structure moving from the periphery of the cell to the Golgi area, and filled arrowheads indicate a tubular structure elongating from the Golgi. Empty arrows and empty arrowheads indicate the initial position of mobile structures. Bars, 5 μm. (D-F) The number of tubule-vesicular structures moving centrifugally (D), the number of tubular structures elongating from the Golgi (E), or the number of tubule-vesicular structures moving centripetally (F), were quantified from videos corresponding to 180 seconds of imaging. Bar represents the mean + standard deviation of the observed profiles (n = 15). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Imaging, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C. The area indicated by a white dotted-line rectangle in each set of images was bleached with a 488-nm laser set to 100% power. The fluorescence recovery after photobleaching (FRAP) was tracked by laser confocal microscopy with the 488-nm laser set to 2% power. Images were acquired before bleaching ( Pre-bleaching ), immediately after bleaching ( Bleaching ), and during the recovery of the fluorescence ( Recovery ) at approximately every 0.4-sec. Images of a representative experiment performed on each cell line are shown in each set of panels. Two images of the recovery of fluorescence are depicted with the time indicated in parenthesis in seconds. Bar, 10 μm. (D) Plot of the FRAP analysis of GFP-GOLPH3 in MCF 10A (black circles; n = 10), MDA-MB-231 (white circles; n = 10), and MCF7 (white squares; n = 10) cells. P , pre-bleaching; B ; bleaching. For simplicity, error bars are not depicted. *** P < 0.001; ns , not statistically significant. The halftime ( t 1/2 ) of maximal fluorescence recovery is indicated on the right in seconds (s).

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Confocal Microscopy

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: Samples (30 μg of proteins) of rat liver cytosol ( Cyt ), rat liver Golgi membranes, and of cytosolic ( Cyt ) and membrane ( Memb ) fractions from the cell lines indicated at the right were analyzed by two-dimensional gel electrophoresis (2-D GE) and immunoblotting using antibody to GOLPH3. Samples of rat liver Golgi membranes, and of the cytosolic and membrane fractions of each cell line, were dephosphorylated with calf intestine alkaline phosphatase ( CIAP ) before processing for 2-D GE. The position of molecular mass markers is indicated on the left. The position of isoelectric point ( pI ) markers is indicated at the bottom. Red asterisks indicate the position of additional, less abundant, but distinct spots in the samples of MCF7 cells that have slightly slower electrophoretic mobility. Numbers indicate different acidic forms identified in immunoblot films subjected to different exposure times.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Membranes with the spotted phospholipids indicated on the left were incubated with untreated, recombinant GOLPH3 ( GOLPH3 ) or with recombinant GOLPH3 in the presence of cytosolic proteins from the cell lines indicated on the top. Bound recombinant GOLPH3 was detected by immunoblotting with antibody to GOLPH3. LysoPtdA , lysophosphatidic acid; LysoPtdCho , lysophosphatidylcholine; PtdIns , phosphatidylinositol; PtdIns(3)P , phosphatidylinositol 3-phosphate; PtdIns(4)P , phosphatidylinositol 4-phosphate; PtdIns(5)P , phosphatidylinositol 5-phosphate; PtdEth , phosphatidylethanolamine; PtdCho , phosphatidylcholine; S1P , sphingosine 1-phosphate; PtdIns(3 , 4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3 , 5)P 2 , phosphatidylinositol 3,5-bisphosphate; PtdIns(4 , 5)P 2 , phosphatidylinositol 4,5-bisphosphate; PtdIns(3 , 4 , 5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PtdA , phosphatidic acid; PtdSer , phosphatidylserine; Blank , no lipid. (B) Densitometric quantification of the immunoblot signal of the levels of untreated, recombinant GOLPH3 bound to different phospholipids as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of recombinant GOLPH3 bound to phosphatidylinositol 4-phosphate after incubation with cytosolic proteins of the indicated cell lines as shown in (A). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Incubation, Recombinant, Western Blot

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 1. Comparison of GOLPH3 mRNA expression in the control and transfection groups. GOLPH3 expression was normalized to 18s rRNA expression and analyzed using Student's t-test. **P<0.01 vs. the control group. GOLPH3, Golgi phosphorylated protein 3; si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison, Expressing, Control, Transfection

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 2. Comparison of GOLPH3 protein expression in the control and transfection groups. GAPDH expression was used to normalize GOLPH3 expression, and Student's t-test was used for analysis. ***P<0.001 vs. the con- trol group. GOLPH3, Golgi phosphorylated protein 3. si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison, Expressing, Control, Transfection

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 3. Comparison of the proliferation of colon cancer cells in the trans- fection and control groups. Student's t-test was used for analysis. **P<0.01 vs. the control group. GOLPH3, Golgi phosphorylated protein 3; OD, optical density; si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison, Control

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 4. Comparison of the number of HT29 colonies between the transfec- tion and control groups. Student's t-test was used for analysis. *P<0.05 vs. the control group. GOLPH3, Golgi phosphorylated protein 3; si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison, Control

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 5. Comparison of apoptosis rates in the transfection and control groups. Student’s t-test was used for analysis. **P<0.01 vs. the control group. GOLPH3, FITC, fluorescein isothiocyanate; Golgi phosphorylated protein 3, PI, propidium iodide; si, small-interfering. Figure 6. Effects of cisplatin on the expression of GOLPH3, P-gp, β-catenin and pERK1/2 proteins. (A) Protein expression following treatment with dif- ferent concentrations of cisplatin. (B) Comparison of protein expression levels following treatment with different concentrations of cisplatin. GAPDH was used for normalization, and expression levels were analyzed by one-way anal- ysis of variance. NS, P>0.05, **P<0.01 and ***P<0.001 vs. the control group. GOLPH3, Golgi phosphorylated protein 3; P-gp, P-glycoprotein; ERK, extra- cellular signal-regulated kinase; NS, non-significant; p, phosphorylated.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison, Transfection, Control, Expressing

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 9. Comparison of tumorigenicity of colon cancer cells under cis- platin treatment. (A) Tumor sphere of HT29 cells in serum-free medium. Magnification, x200. (B) Comparison of tumor sphere counts in each group. Least Significant Difference t-test was used for analysis. NS, P>0.05 and ***P<0.001. GOLPH3, Golgi phosphorylated protein 3; NS, non-significant; si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 7. Inhibitory effect of cisplatin on the proliferation of HT29 cells. Least Significant Difference t-test was used for analysis. *P<0.05 and ***P<0.001. GOLPH3, Golgi phosphorylated protein 3; OD, optical density; si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques:

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 8. Comparison of the numbers of HT29 colonies under cisplatin treatment. (A) Images of cell colonies in each group. (B) Comparison of the number of colonies in each group. Least Significant Difference t-test was used for analysis. **P<0.01 and ***P<0.001. GOLPH3, Golgi phosphorylated protein 3; OD, optical density; si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 11. Protein expression in experimental groups 1 and 2 under cis- platin treatment. (A) Protein expression bands. (B) Comparison of protein expression in each group. Least Significant Difference t-test was used for analysis, and GAPDH was used for normalization. NS, P>0.05, *P<0.05, **P<0.01 and ***P<0.001. GOLPH3, Golgi phosphorylated protein 3; P-gp, P-glycoprotein; ERK, extracellular signal-regulated kinase; NS, non-signifi- cant; si, small‑interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Expressing, Comparison

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 10. Comparison of the apoptosis of HT29 cells under cisplatin treatment in different groups. (A) Analysis of apoptosis of HT29 cells. (B) Comparison of apoptosis rates in each group. Least Significant Difference t-test was used for analysis. **P<0.01 and ***P<0.001. FITC, fluorescein iso- thiocyanate; GOLPH3, Golgi phosphorylated protein 3; PI, propidium iodide; si, small-interfering.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison

Journal: International journal of oncology

Article Title: Silencing GOLPH3 gene expression reverses resistance to cisplatin in HT29 colon cancer cells via multiple signaling pathways.

doi: 10.3892/ijo.2018.4471

Figure Lengend Snippet: Figure 13. Comparison of the volume of subcutaneous tumors in nude mice in each group. (A) Subcutaneous tumor specimens from the nude mice in each group. (B) Growth curve of subcutaneous transplanted tumor in nude mice in each group. GOLPH3, Golgi phosphorylated protein 3; si, small‑interfering. ***P<0.001.

Article Snippet: The blots were incubated with antibodies against GOLPH3 (1:1,000; catalog no. ab 98023); P-glycoprotein (p-gp; 1:500; catalog no. ab 103477) (both from Abcam, Cambridge, UK); β-catenin (1:500; catalog no. sc-59737; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 (1:500; catalog no. ab 17942); pERK1/2 (1:500; catalog no. ab 65142) (both from Abcam); GAPDH (1:1,000; catalog no. sc-365062); and β-actin (1:1,000; catalog no. sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4 ̊C overnight.

Techniques: Comparison

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: 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: The Febs Journal

Article Title: Involvement of GTPases and vesicle adapter proteins in Heparan sulfate biosynthesis: role of Rab1A , Rab2A and GOLPH3

doi: 10.1111/febs.17398

Figure Lengend Snippet: 3OST5 interacts with GOLPH3. EC‐3OST5 cell lysate was immunoprecipitated for GFP using the anti‐GFP antibody and revealed for the GOLPH3 protein. The upper membrane was incubated with the primary antibody against GOLPH3 (1 : 1000) and detected using an anti‐rabbit HRP‐linked secondary antibody (1 : 2500). Meanwhile, the lower membrane was developed using only an anti‐goat HRP‐linked secondary antibody (1 : 2500) and, as a result, the band corresponding to the 3OST5‐GFP protein in the input lane is absent. The data reflect the outcomes of two independent experiments.

Article Snippet: Rabbit antibodies against α‐COP, β‐COP, GM130, Calreticulin and Syndecan‐3 were purchased from Abcam, antibodies against 3OST5 and COPII from Thermo Scientific (Rockford, IL, USA), antibody to 2OST (N‐term) from Abgent, antibodies against Rab1A and GOLPH3 from Origene and antibodies against 6OST1, Syndecan‐2 and Syndecan‐4 from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunoprecipitation, Membrane, Incubation