Journal: bioRxiv

Article Title: Phage display-mediated immuno-PCR to detect low-abundance secreted proteins in Drosophila

doi: 10.1101/2025.10.31.685841

Figure Lengend Snippet: ImpL2 nanobody screening using phage-displayed nanobody library. (A) Schematic of ImpL2 nanobody screening. A Maxisorp plate coated with mCherry-hIgG was used for negative selection, and a coated plate with ImpL2-hIgG was used for positive selection. Three iterative rounds of selection were conducted. (B) ELISA results using polyclonal phages from three rounds of selection. The coating amount of mCherry-hIgG and ImpL2-hIgG decreased across the rows. The intensity of the green color indicates the strength of the ELISA signal. (C) ELISA results of monoclonal phages isolated after the third round of selection. 96 individual clones were tested against the control protein (mCherry-hIgG) and the target protein (ImpL2-hIgG). (D) Immunostaining of cells expressing membrane-tethered ImpL2 using nanobody-displaying phages. GFP signals mark cells expressing membrane-tethered ImpL2. Bound phages were detected using an antibody against the M13 major coat protein. Nb.b201-displaying phage was used as a negative control. BF, bright field. (E) Immunostaining of cells expressing membrane-tethered ImpL2 using recombinant nanobodies. GFP signals mark cells expressing membrane-tethered ImpL2. Bound nanobodies were detected using an antibody against alpaca VHH fragments. Recombinant Nb127D01 was used as a negative control. BF, bright field.

Article Snippet: Other antibodies used in this study include anti-M13 major coat protein antibody (Santa Cruz Biotechnology, sc-53004 HRP and sc-53004 AF647), anti-alpaca VHH domain antibody (Jackson ImmunoResearch, 128-605-230 and 128-035-230) and anti-Human IgG Fc antibody (Thermo Fisher Scientific, A18829).

Techniques: Selection, Enzyme-linked Immunosorbent Assay, Isolation, Clone Assay, Control, Immunostaining, Expressing, Membrane, Negative Control, Recombinant

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