Journal: European Journal of Histochemistry : EJH

Article Title: SRSF3 promotes the generation of XBP1s to stabilize autophagy and enhance hypoxia adaptation in glioma

doi: 10.4081/ejh.2026.4530

Figure Lengend Snippet: Hypoxic glioma cells exhibit increased cell death accompanied by elevated endoplasmic reticulum stress levels and blocked autophagic flux. A ) Western blot analysis of HIF-1α expression in U87 cells. B ) LDH release assay to detect cell death. C ) Western blot analysis of cleaved-caspase3 expression in U87 cells. D ) Western blot analysis of GRP78, CHOP, and ATF4 expression in U87 cells. E ) RT-PCR detection of XBP1u and XBP1s expression. F ) Western blot analysis of XBP1u and XBP1s expression in U87 cells. G ) Western blot analysis of LC3-II and p62 expression in U87 cells. H ) mRFP-GFP-LC3 staining to assess autophagic flux. n=3 independent experiments; * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The primary antibodies used were against the following: SRSF3 (1:1000; ab198291; Abcam, Cambridge, UK), HIF-1α (1:1000; ab179483; Abcam), GRP78 (1:2000; 11587-1-AP; Proteintech, Rosemont, IL, USA), CHOP (1:1000; 15204-1-AP; Proteintech), ATF4 (1:1000; 10835-1-AP; Proteintech), LC3 (1:2000; ab192890; Abcam), p62 (1:1000; ab109012; Abcam), cleaved-caspase3 (1:1000; #9661; Cell Signaling Technology, Inc., Danvers, MA, USA), XBP1u (1:1000; 25997-1-AP; Proteintech), XBP1s (1:1000; 24868-1-AP; Proteintech), and β-actin (1:1000; ab8227; Abcam).

Techniques: Western Blot, Expressing, Lactate Dehydrogenase Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: SRSF3 promotes the generation of XBP1s to stabilize autophagy and enhance hypoxia adaptation in glioma

doi: 10.4081/ejh.2026.4530

Figure Lengend Snippet: SRSF3 promotes XBP1s formation. A ) RT‒PCR detection of XBP1u and XBP1s expression in each group of cells. B ) Western blot detection of XBP1u and XBP1s expression in each group of cells. n=3 independent experiments; * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The primary antibodies used were against the following: SRSF3 (1:1000; ab198291; Abcam, Cambridge, UK), HIF-1α (1:1000; ab179483; Abcam), GRP78 (1:2000; 11587-1-AP; Proteintech, Rosemont, IL, USA), CHOP (1:1000; 15204-1-AP; Proteintech), ATF4 (1:1000; 10835-1-AP; Proteintech), LC3 (1:2000; ab192890; Abcam), p62 (1:1000; ab109012; Abcam), cleaved-caspase3 (1:1000; #9661; Cell Signaling Technology, Inc., Danvers, MA, USA), XBP1u (1:1000; 25997-1-AP; Proteintech), XBP1s (1:1000; 24868-1-AP; Proteintech), and β-actin (1:1000; ab8227; Abcam).

Techniques: Expressing, Western Blot

Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation of NS3/NS3A facilitates its raft-enriched membrane association and efficient BTV release. ( A ) Representative confocal images of HeLa cells transfected with NS3/NS3A WT or the N150Q mutant. Cells were fixed and stained 20 h post-transfection. NS3/NS3A was detected using anti-NS3/NS3A (red), and lipid raft-enriched membrane domains were labeled with anti-flotillin-1 (green). Scale bars, 5 μm. ( B and C ) Confocal images of HeLa cells expressing NS3/NS3A treated with trifluoperazine (TFP) ( B ) or methyl-β-cyclodextrin (MβCD) ( C ). Cells were fixed and stained 20 h post-transfection. NS3/NS3A is shown in red and flotillin-1 in green. Fluorescence intensity profiles were analyzed along the indicated line scans. Scale bars, 5 μm. ( D ) Localization of NS3/NS3A at the plasma membrane of HeLa cells with or without MβCD or TFP treatment. The plasma membrane was stained using WGA-Alexa Fluor 488 (green), and NS3/NS3A was visualized in red. Scale bars, 5 μm. ( E ) Quantification of plasma membrane (PM) localization from panel D, shown as the ratio of PM-associated NS3/NS3A fluorescence intensity (defined by WGA staining) to total cellular NS3/NS3A intensity. Values were normalized to vehicle-treated controls and are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (** P < 0.01, **** P < 0.0001). ( F ) MDOK or HEK-293T cells were infected with BTV-20 WT or BTV-20 N150Q at an MOI of 5. At 6 h post-infection, the cells were treated with MβCD or TFP, and viral titers were determined at 12 h post-infection. Data represent mean ± SD ( n = 3 biological replicates). Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; *** P < 0.001; **** P < 0.0001). ( G ) Analysis of BTV-20 release efficiency following raft disruption. MDOK cells were infected with BTV-20 (MOI = 5) and then treated with MβCD or TFP. Extracellular and intracellular viral titers were determined at the indicated time points. Release efficiency was calculated as [extracellular titer/(extracellular + intracellular titer)] × 100%. Data are presented as mean ± SD ( n = 3 biological replicates). Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). ( H ) A total of 1,136 host proteins were identified by quantitative proteomics, of which 102 proteins were enriched in the NS3/NS3A WT interactome relative to the N150Q mutant (log₂ FC > 4 or unique to WT). Highlighted are the top eight candidates. ( I ) Gene Ontology (GO) enrichment analysis of proteins enriched in the NS3/NS3A WT interactome. Significantly overrepresented terms are shown for the biological process, cellular component, and molecular function categories. Bars represent −log₁₀ ( P value). ( J ) Sucrose gradient fractionation followed by immunoblotting of lysates from transfected HEK-293T cells, or from infected MDOK cells. DRM (raft-enriched) fractions correspond to fractions 5–12, whereas detergent-soluble membrane (DSM) fractions correspond to fractions 13–20. ( K ) Co-immunoprecipitation analysis of NS3/NS3A WT , NS3/NS3A N150Q , and FLNA. Cells were transfected with NS3/NS3A variants, followed by Co-IP.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Membrane, Transfection, Mutagenesis, Staining, Labeling, Expressing, Fluorescence, Clinical Proteomics, Infection, Disruption, Quantitative Proteomics, Fractionation, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Journal of Proteomics

Article Title: Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

doi: 10.1016/j.jprot.2012.06.024

Figure Lengend Snippet: Verification of protein expression and localization in synovial fluid and membrane. A. Representative MALDI-TOF mass spectra of spot 873 identified as vitamin D-binding protein. The matched peptide sequences are underlined within the sequence of vitamin D-binding protein. An approximate molecular weight of the protein cleavage product (between the arrows) is calculated from the amino acid sequence, in line with DIGE estimate. B. Immunoprecipitation of VDBP from synovial fluids of n = 48 representative JIA patients (indicated by number in lanes 1–48; M — molecular weight marker; D — purified human VDBP positive control). Band densities of vitamin D binding protein concur with protein expression levels measured by DIGE (extended-to-be oligoarticular patients in lanes 13, 21, 24, 36, 37, 45, 47, 48). C. Representative immunohistochemistry of neighboring sections of synovial membrane from a polyarticular patient, all captured at 10 × magnification. Vitamin D binding protein expression is perivascular in nature. D. ELISA quantification of c-reactive protein and vitamin D binding protein concentration in initial plasma and synovial samples taken from the whole study cohort. Error bars on the box-whisker plots represent range between maximum and minimum values; center line represents median.

Article Snippet: A purified form of human VDBP was run with IP samples as a positive control (2 μg) (Athens Research & Technology Inc., Athens, GA, USA).

Techniques: Expressing, Membrane, Binding Assay, Sequencing, Molecular Weight, Immunoprecipitation, Marker, Purification, Positive Control, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Protein Concentration, Clinical Proteomics, Whisker Assay