Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Analysis of de novo RBD expression, secretion, and intracellular TEVp-mediated cleavage in co-transfected HEK293T cells (A) Overview of the experimental set-up to analyze the total RBD-V5-His release and TEVp- c -Myc-His secretion (left side) and de novo RBD-V5-His release (right side) from co-transfected HEK293T cells. (B) Western blot analysis of the samples derived from experiment (A) by detection of V5 or c -Myc tag. (C) Western blot analysis of the secreted and residual intracellular RBD-V5-His (top) and TEVp N23Q,C130S,T173G,S219V - c -Myc-His (bottom) isolated using His-tag from 1 mL of supernatant (left lanes) or lysed co-transfected HEK293T cells from one well of a 6-well plate in 1 mL (right lanes). Cells were lysed with 1% of IGEPAL CA-630, which was also added to the supernatant. (D) Coomassie Brilliant blue-stained polyacrylamide gel quantified by comparison with a bovine serum albumin standard curve (left) and subsequent western blot probed by anti-V5 antibodies (right) of the protein released from the supernatant of co-transfected HEK293T cells obtained by co-expression of tANCHORed RBD-V5-His (top) or CD63LEL-V5-His (bottom) with or without TEVp N23Q,C130S,T173G,S219V - c -Myc-His. Highly glycosylated CD63LEL-V5-His was treated with PNGase F (peptide N-glycosidase F). (E) Lectin blot of the released RBD-V5-His protein, probed with biotinylated Sambucus nigra agglutinin (SNA) and detected using streptavidin-HRP. (F and G) Western blot analysis of the cell lysates for the expression of CD63-mCherry (F) or CD82-tANCHOR-CD63LEL-mCherry (G), with or without modified TEVp expression. Control contains untransfected cells, and TEVp was captured from the supernatant.

Article Snippet: After blocking, the membrane was washed and incubated for 2 h with biotinylated Sambucus nigra lectin (SNA, Vector Laboratories, Biozol, Hamburg, Germany) at a concentration of 5 μg/mL in 3% BSA.

Techniques: Expressing, Transfection, Western Blot, Derivative Assay, Isolation, Staining, Comparison, Modification, Control

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Analysis of de novo RBD expression, secretion, and intracellular TEVp-mediated cleavage in co-transfected HEK293T cells (A) Overview of the experimental set-up to analyze the total RBD-V5-His release and TEVp- c -Myc-His secretion (left side) and de novo RBD-V5-His release (right side) from co-transfected HEK293T cells. (B) Western blot analysis of the samples derived from experiment (A) by detection of V5 or c -Myc tag. (C) Western blot analysis of the secreted and residual intracellular RBD-V5-His (top) and TEVp N23Q,C130S,T173G,S219V - c -Myc-His (bottom) isolated using His-tag from 1 mL of supernatant (left lanes) or lysed co-transfected HEK293T cells from one well of a 6-well plate in 1 mL (right lanes). Cells were lysed with 1% of IGEPAL CA-630, which was also added to the supernatant. (D) Coomassie Brilliant blue-stained polyacrylamide gel quantified by comparison with a bovine serum albumin standard curve (left) and subsequent western blot probed by anti-V5 antibodies (right) of the protein released from the supernatant of co-transfected HEK293T cells obtained by co-expression of tANCHORed RBD-V5-His (top) or CD63LEL-V5-His (bottom) with or without TEVp N23Q,C130S,T173G,S219V - c -Myc-His. Highly glycosylated CD63LEL-V5-His was treated with PNGase F (peptide N-glycosidase F). (E) Lectin blot of the released RBD-V5-His protein, probed with biotinylated Sambucus nigra agglutinin (SNA) and detected using streptavidin-HRP. (F and G) Western blot analysis of the cell lysates for the expression of CD63-mCherry (F) or CD82-tANCHOR-CD63LEL-mCherry (G), with or without modified TEVp expression. Control contains untransfected cells, and TEVp was captured from the supernatant.

Article Snippet: Sambucus nigra lectin (SNA, EBL) biotinylated , Vector Laboratories , Cat# B-1305-2.

Techniques: Expressing, Transfection, Western Blot, Derivative Assay, Isolation, Staining, Comparison, Modification, Control

Journal: Infection and Immunity

Article Title: The growth and pathogenesis of Citrobacter rodentium are compromised when multiple mucin sugar utilization pathways are disrupted, leading to accumulation of N-acetylglucosamine 6-phosphate

doi: 10.1128/iai.00545-25

Figure Lengend Snippet: C. rodentium utilizes GlcNAc and NeuNAc that are enriched in the murine colon of C57BL/6J mice. ( A ) Growth assay of WT C. rodentium in minimal media with five monosaccharides constituting the Muc2 O-glycans. The absorbance at OD 600 was measured every hour and was shown as mean ± SEM from biological triplicates. ( B ) Wheat germ agglutinin (WGA) and ( C ) Sambucus nigra agglutinin (SNA) staining of colonic cross sections from uninfected mice (at baseline). Sections were stained with WGA or SNA (green) to detect GlcNAc or NeuNAc, respectively, and 4′,6′-diamidino-2-phenylindole (DAPI) (blue) to detect DNA. Original magnification, 200×. Images are representative of three independent experiments with five mice per experiment (scale bar, 20 μm). ( D ) Levels of GlcNAc and NeuNAc in the stools of Muc2 +/+ and Muc2 −/− littermates assessed by an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ-MS). Data were collected from 10 mice pooled from two independent experiments and shown as mean ± SEM. Statistical significance was determined by multiple Mann-Whitney tests. **** P < 0.0001, *** P < 0.001. Levels of ( E ) GlcNAc and ( F ) NeuNAc in the stool samples of mice at baseline and during infection with C. rodentium were assessed by UHPLC/QqQ-MS. Data were collected from eight mice pooled from three independent experiments and shown as mean ± SEM. Statistical significance was determined by Kruskal-Wallis tests (E and F). * P < 0.05. The Y-axis represents the amount of sugar in micrograms per gram of stool ( D–F ).

Article Snippet: Paraffin-embedded colonic tissue sections (5 μm) were deparaffinized by heating at 60°C for 15 min, cleared with xylene, and rehydrated with 100%, 95%, and 70% ethanol, followed by dH 2 O. Dewaxed and dehydrated colonic tissue sections were blocked with Donkey Serum buffer at room temperature (RT) for 1 h. Fluorescently labeled lectins were diluted in antibody dilution buffer and used for staining: WGA (Catalog #FL-1021, Vector Laboratories, 1:500) for GlcNAc, and SNA (Catalog #FL-1301, Vector Laboratories, 1:200) for NeuNAc.

Techniques: Growth Assay, Staining, High Performance Liquid Chromatography, Targeted Proteomics, Mass Spectrometry, MANN-WHITNEY, Infection