Journal: Scientific reports

Article Title: GOLPH3 promotes calcium oxalate-induced renal injury and fibrosis through Golgi stress-mediated apoptosis and inflammatory responses.

doi: 10.1038/s41598-025-91638-0

Figure Lengend Snippet: Fig. 1. Up-regulation of GOLPH3 expression in renal tissues of patients with kidney stones (A) Representative Tunel staining results of normal renal tissue and renal tissue from patients with renal stones (n = 6). (B, C, E) IHC, qPCR and Western blot analyses of GOLPH3 expression levels in nephrolithiasis patients. (D) Online single-cell sequencing database of snRNA-seq validates GOLPH3 expression levels in normal kidneys versus kidneys of AKI and CKD patients. (F) Representative images (n = 6) of immunofluorescence staining for GOLPH3 (green) and GM130 (red). Data are the mean ± SEM of at least 3 independent experiments. *P < 0.05 , ***P < 0.001 versus NC group.

Article Snippet: These sections were then incubated overnight at 4 °C with anti-GOLPH3 antibody (1:500; Proteintech, Wuhan, China), followed by incubation with horseradish peroxidase (HRP)-conjugated goat secondary antibody at 37 °C for 1 h. The following day, the sections were visualized using 3,3′-diaminobenzidine (DAB).

Techniques: Expressing, TUNEL Assay, Staining, Western Blot, Sequencing, Immunofluorescence

Journal: Scientific reports

Article Title: GOLPH3 promotes calcium oxalate-induced renal injury and fibrosis through Golgi stress-mediated apoptosis and inflammatory responses.

doi: 10.1038/s41598-025-91638-0

Figure Lengend Snippet: Fig. 2. Glyoxalate-induced stone model promotes renal fibrosis and inflammatory response (A) Diagram depicting the experimental design. Measurement of the levels of BUN and Scr in the serum of mice (n = 6). (C) mRNA expression of KIM‑1 and NGAL in mice kidney tissue. (D) Serum levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were quantified with ELISA. (E) Representative images of HE, Von Kossa, Masson, Sirius red, Tunel staining and immunohistochemical staining for GOLPH3 of mouse kidney tissue (n = 6). (F, G) Western blot and qPCR analyses of GOLPH3 expression levels in mice kidney. Data are the mean ± SEM of at least three independent experiments. ****P < 0.0001 versus NC group.

Article Snippet: These sections were then incubated overnight at 4 °C with anti-GOLPH3 antibody (1:500; Proteintech, Wuhan, China), followed by incubation with horseradish peroxidase (HRP)-conjugated goat secondary antibody at 37 °C for 1 h. The following day, the sections were visualized using 3,3′-diaminobenzidine (DAB).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Immunohistochemical staining, Western Blot

Journal: Scientific reports

Article Title: GOLPH3 promotes calcium oxalate-induced renal injury and fibrosis through Golgi stress-mediated apoptosis and inflammatory responses.

doi: 10.1038/s41598-025-91638-0

Figure Lengend Snippet: Fig. 4. Deposited calcium oxalate stones regulates the PI3K/AKT/mTOR signaling in HK-2 cells (A) Western blots assay was used to detect the expressions of GOLPH3, Golgi structure and functional proteins (GRASP65, ARF4, PI4KIIIβ and MYO18A) and proteins related to renal fibrosis (Collagen I, Vimentin, α-SMA) in HK-2 cells. (B) Representative images of immunofluorescence staining of HK-2 cells GOLPH3 (red) and GM130 (green) (C) Expressions of PI3K/AKT/mTOR pathway-related proteins in HK-2 cells quantified by Western blotting. Data are the mean ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001 versus NC group.

Article Snippet: These sections were then incubated overnight at 4 °C with anti-GOLPH3 antibody (1:500; Proteintech, Wuhan, China), followed by incubation with horseradish peroxidase (HRP)-conjugated goat secondary antibody at 37 °C for 1 h. The following day, the sections were visualized using 3,3′-diaminobenzidine (DAB).

Techniques: Western Blot, Functional Assay, Immunofluorescence, Staining

Journal: Scientific reports

Article Title: GOLPH3 promotes calcium oxalate-induced renal injury and fibrosis through Golgi stress-mediated apoptosis and inflammatory responses.

doi: 10.1038/s41598-025-91638-0

Figure Lengend Snippet: Fig. 5. Inhibition of GOLPH3 reduces apoptosis and inflammatory responses in Golgi stress-mediated HK-2 cells (A) Confirmation of siRNA-mediated GOLPH3 knockdown efficiency using immunoblotting in HK-2 cells. (B) The levels of TNF-α, IL-6, and IL-1β were determined by qPCR. (C, D) The expression of GOLPH3, Golgi structural and functional proteins (GRASP65, ARF4, PI4KIIIβ, and MYO18A) and renal fibrosis-related proteins (collagen I, waveform protein, and α-SMA) were detected by Western blotting and qRT-PCR in HK-2 cells in each group. (E) After treating HK-2 cells with 200 μg/mL COM for 24 h, the proportion of apoptotic HK-2 cells in each group was assessed by flow cytometry. (F) Expression of GOLPH3 in HK-2 cell was detected by immunofluorescence staining, and the percent of positive cells was quantified. (G) Western blotting was used to quantify the expression of PI3K/AKT/mTOR pathway-related proteins in HK-2 cells of each group. Data are the mean ± SEM of at least three independent experiments. **P < 0.01, ***P < 0.001,****P < 0.0001 versus Ctrl-siRNA group. #P < 0.01, ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus Ctrl-siRNA + COM group.

Article Snippet: These sections were then incubated overnight at 4 °C with anti-GOLPH3 antibody (1:500; Proteintech, Wuhan, China), followed by incubation with horseradish peroxidase (HRP)-conjugated goat secondary antibody at 37 °C for 1 h. The following day, the sections were visualized using 3,3′-diaminobenzidine (DAB).

Techniques: Inhibition, Knockdown, Western Blot, Expressing, Functional Assay, Quantitative RT-PCR, Flow Cytometry, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin

doi: 10.1016/j.jbc.2021.101067

Figure Lengend Snippet: Commercial antibodies used in this study

Article Snippet: M13 RL-ph1 ∗HRP , Santa Cruz Cat# sc-53004 RRID: AB_673750 , M13 phage coat protein (monoclonal∗HRP).

Techniques: Immunostaining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer

doi: 10.1186/s13046-018-0808-1

Figure Lengend Snippet: STK25 interacts with GOLPH3 and regulates its expression. a , b Exogenous STK25 interacts with GOLPH3. Cells were transfected with the indicated plasmids. Co-IP was performed using FLAG antibody to pull down FLAG-STK25 ( a ) or anti-Myc against Myc-GOLPH3 ( b ). Then, STK25 and GOLPH3 were detected with the indicated antibodies. c , d His-STK25 interacts directly with GST-GOLPH3 but not GST by in vitro GST pull-down and His-tag pull-down assays, respectively. e STK25 overexpression decreases GLOPH3 mRNA and protein levels in CRC cells. f STK25 knockdown increases GOLPH3 mRNA and protein levels in CRC cells

Article Snippet: The primary antibodies were used at the following dilutions: STK25 (1:1000, Cat #25821–1-AP, Proteintech), GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech), PDHK1 (1:1000, Cat #3820, Cell Signaling Technology), HK2 (1:1000, Cat #2867, Cell Signaling Technology), LDHA (1:1000, Cat #2012, Cell Signaling Technology), AKT (1:1000, Cat #9272, Cell Signaling Technology), p-AKT (1:1000, Cat #9271, Cell Signaling Technology), mTOR Substrates Antibody Sampler Kit (1:1000, Cat #9862, Cell Signaling Technology), p70S6K (1:1000, Cat #2708, Cell Signaling Technology), p-p70S6K (1:200, Cat #9234, Cell Signaling Technology), and GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech).

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, In Vitro, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer

doi: 10.1186/s13046-018-0808-1

Figure Lengend Snippet: STK25 modulates glycolysis via GOLPH3. Culture media from controls and GOLPH3-overexpressing or GOLPH3-depleted CRC cells were analyzed to measure relative glucose uptake ( a ), lactate production ( b ), and lactate:glucose ratio ( c ). Knockdown of GOLPH3 decreases the promotion of glucose uptake ( d ) and lactate production ( e ) induced by STK25 depletion in LoVo cells. Data are expressed as mean ± SD. *, P < 0.05

Article Snippet: The primary antibodies were used at the following dilutions: STK25 (1:1000, Cat #25821–1-AP, Proteintech), GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech), PDHK1 (1:1000, Cat #3820, Cell Signaling Technology), HK2 (1:1000, Cat #2867, Cell Signaling Technology), LDHA (1:1000, Cat #2012, Cell Signaling Technology), AKT (1:1000, Cat #9272, Cell Signaling Technology), p-AKT (1:1000, Cat #9271, Cell Signaling Technology), mTOR Substrates Antibody Sampler Kit (1:1000, Cat #9862, Cell Signaling Technology), p70S6K (1:1000, Cat #2708, Cell Signaling Technology), p-p70S6K (1:200, Cat #9234, Cell Signaling Technology), and GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer

doi: 10.1186/s13046-018-0808-1

Figure Lengend Snippet: STK25 mediates glycolysis through GOLPH3-regulated mTOR signaling. Western blot was performed to assess the levels and phosphorylation of mTOR substrates in LoVo cells transfected with STK25 expression plasmid ( a ) or STK25 siRNA ( b ). c Knockdown of GOLPH3 partially reverses the upregulation of pAKT and pS6K induced by STK25 depletion. The relative phosphorylation levels of specific substrates of mTORC1 and mTORC2 were quantified by assessing the relative intensity of phosphorylated bands to corresponding protein bands ( a - c ; shown as mean ± SD of three scans). Rapamycin attenuates STK25 knockdown-induced glucose uptake ( d ) and lactate production ( e ). Data are expressed as mean ± SD. *, P < 0.05

Article Snippet: The primary antibodies were used at the following dilutions: STK25 (1:1000, Cat #25821–1-AP, Proteintech), GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech), PDHK1 (1:1000, Cat #3820, Cell Signaling Technology), HK2 (1:1000, Cat #2867, Cell Signaling Technology), LDHA (1:1000, Cat #2012, Cell Signaling Technology), AKT (1:1000, Cat #9272, Cell Signaling Technology), p-AKT (1:1000, Cat #9271, Cell Signaling Technology), mTOR Substrates Antibody Sampler Kit (1:1000, Cat #9862, Cell Signaling Technology), p70S6K (1:1000, Cat #2708, Cell Signaling Technology), p-p70S6K (1:200, Cat #9234, Cell Signaling Technology), and GOLPH3 (1:1000, Cat #19112–1-AP, Proteintech).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) HeLa S3 cells were uninfected or infected with HPV16.L2F (MOI, ~100). PLA (green) for IPO7 and FLAG (to detect L2) was performed at 24 hpi; nuclei were stained with DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( B ) PLA fluorescence intensity per cell (>100 cells) as in (A), shown as individual values, means, and SDs, with statistical significance determined by one-way ANOVA. **** P < 0.0001. ( C ) Whole-cell extracts from uninfected or HPV16.L2F-infected (MOI, ~1) HeLa cells were collected at the indicated time points. A portion of the resulting extracts (input) was analyzed by immunoblotting for FLAG (to detect L2) and IPO7. The remaining extracts were subjected to IP with an anti-IPO7 antibody or control IgG and analyzed by immunoblotting for FLAG and IPO7. ( D ) HeLa cells were pretreated with 9 μM RO3306 or DMSO as a control for 24 hours and then uninfected or infected with HPV16.L2F (MOI, ~1), with RO3306 or DMSO maintained in the medium. At 24 hpi, cells were lysed and analyzed in (C). ( E ) HeLa cells treated with 10 nM Scr or a mixture of 5 nM COPA and 5 nM COPG1 ( COPA/G1 ) siRNA were uninfected or infected with HPV16.L2F PsV (MOI, ~1). At 24 hpi, cells were lysed, and a portion of the extracts (input) was immunoblotted for FLAG, IPO7, γ-COP, or β-actin (as a loading control). The remaining extracts were subjected to IP with an anti-IPO7 antibody or a control IgG and analyzed as in (C). ( F ) As in (E), except cells were transfected with 10 nM Scr or IPO7 siRNA #2 before infection. Samples were subjected to IP with an anti–γ-COP antibody or control IgG and analyzed as in (E).

Article Snippet: γ-COP , Rabbit , 12393-1-AP; Proteintech , WB and IP.

Techniques: Infection, Staining, Fluorescence, Western Blot, Control, Transfection

Journal: Veterinary Research

Article Title: Comprehensive multiomic analysis of extracellular vesicles from Mycoplasma bovis -infected bovine mammary epithelial cells identifies proteins and miRNAs that induce inflammatory responses in macrophages

doi: 10.1186/s13567-025-01626-5

Figure Lengend Snippet: Quantitative proteomic analysis of differentially expressed proteins in Ctrl-EVs and M. bovis NX2-EVs . A Venn diagram showing common and unique proteins in Ctrl-EVs and M. bovis NX2-EVs. B Statistics on the identification and quantification of proteins in Ctrl-EVs and M. bovis NX2-EVs. C Histogram of the subcellular localization of differentially expressed proteins between M. bovis NX2-EVs and Ctrl-EVs. D GO enrichment analysis of all significantly differentially expressed proteins; BP (biological process), CC (cellular component) and MF (molecular function) are indicated by green, blue and red, respectively. E KEGG enrichment analysis of all significantly differentially expressed proteins. F Volcano diagram of differentially expressed proteins between M. bovis NX2-EVs and Ctrl-EVs; red and blue dots indicate significantly upregulated and downregulated proteins, respectively, whereas grey dots represent proteins whose expression was not significantly different. G The abundance of the JCHAIN, JAG1, MAPRE1, ARCN1, APLP2, TSG101 and CD63 proteins was determined by western blotting. H Representative TEM of EVs labelled with immunogold using anti-JCHAIN, anti-JAG1, anti-MAPRE1, anti-ARCN1 and anti-TSG101 Antibodies. Scale bar: 100 nm.

Article Snippet: The grids were subsequently incubated for 2 h at 4 °C with appropriate dilutions of the primary antibodies against ARCN1 (1:1000, ProteinTech), MAPRE1 (1:1000, Affinity), JAG1 (1:1000, ProteinTech), TSG101 (1:1000, ProteinTech) and JCHAIN (1:1000, AtaGenix), and after repeating the washing step (3 min each time), the grid was floated for 2 h at room temperature on Anti-rabbit IgG, Anti-mouse IgG, and secondary antibody droplets containing 10-nm gold particles (AURION, Hatfield, PA).

Techniques: Expressing, Western Blot