Journal: bioRxiv

Article Title: Sialidases derived from Gardnerella vaginalis remodel the sperm glycocalyx and impair sperm function

doi: 10.1101/2025.02.01.636076

Figure Lengend Snippet: A) Schematic describing lectin flow cytometry and representative glycan epitopes. B, C, D) Sperm were exposed to sialidases (AUS 20 units or GvNanH2 160 units) for 1 hour, fixed in formalin, and stained with MAL-II-biotin/Neutravidin-Alexa Fluor 488, SNA-Cy5, or PSA-FITC respectively. Each point represents the median fluorescence intensity from one donor. Statistics represent a mixed model comparison with post-hoc Holm-Šidák tests. E) Schematic representing change in zeta potential after removal of negatively charged sialic acid on sperm surface. F) Sperm were treated with sialidase at various doses for 1 hour, and zeta potential was measured via dynamic light scattering. Each measurement represents the average of seven technical replicates, and statistics represent a mixed model comparison with post hoc Holm-Šidák tests.

Article Snippet: Biotinylated Maackia Amurensis Lectin II (MAL II, Cat #:B-1265-1) and Cy5-conjugated Sambucus Nigra Lectin (SNA, Cat #: CL-1305-1) were purchased from Vector Labs. Human Contraception Antibody (HCA), was a gift from ZabBio.

Techniques: Flow Cytometry, Staining, Fluorescence, Comparison, Zeta Potential Analyzer

Journal: The Journal of biological chemistry

Article Title: The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

doi: 10.1074/jbc.271.9.4632

Figure Lengend Snippet: FIG. 5. Phenotype of a strain (DccsA) containing a disrupted ccsA gene. Soluble proteins were prepared from copper-supplemented (1Cu) or copper-deficient (2Cu) cultures of the indicated strains and analyzed for the accumulation of holocytochrome c6 (heme stain and anti-cytochrome c6) and plastocyanin (anti-pc). Nitrocellulose membranes were used for the leftmost three panels. Bound antibody was detected by use of a horseradish peroxidase-conjugated secondary antibody. The pellet fractions from the preparation were analyzed for cytochrome f accumulation (anti-cytochrome f). PVDF membranes were used for the transfer, and an alkaline phosphatase-conjugated secondary antibody was used for detection.

Article Snippet: Bound antibodies were detected with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibody according to instructions provided by the manufacturer (Bio-Rad).

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

doi: 10.1074/jbc.271.9.4632

Figure Lengend Snippet: FIG. 6. Complementation of strain B6 with the cloned ycf5 gene. A, sum- mary of the complementation experi- ments. Plasmids pEBP and pEBH were constructed in vector pTZ19R, while pNH was constructed in the vector pET22b(1). The indicated frequencies are the aver- ages from three different experiments. B, extracts of soluble protein were prepared from copper-deficient cultures of strain CC425 (wt), strain B6, or cells of B6 res- cued by plasmid pEBP (B6-P) or pEBH (B6-H). Equivalent amounts (correspond- ing to 1 A595 unit in the Pierce Coomassie dye binding assay) were analyzed, after separation of proteins in an SDS-contain- ing polyacrylamide gel and transfer to a nitrocellulose membrane, for accumula- tion of holocytochrome c6 by heme stain- ing (bottom panel, 45-min exposure to NEN Reflection film) or decoration with anti-cytochrome c6 (top panel, horse- radish peroxidase conjugated second antibody).

Article Snippet: Bound antibodies were detected with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibody according to instructions provided by the manufacturer (Bio-Rad).

Techniques: Clone Assay, Construct, Plasmid Preparation, Binding Assay, Membrane, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 4. Perinuclear Localization of the Aglycosylated PRLR COS-7 cells transfected with WT (a and d), N80,108D (b and e), or N35,80,108D (c and f) PRLR cDNAs were fixed in 4% paraformaldehyde (a, b, and c: nonpermeabilized conditions) or in methanol, 220 C (d, e, and f: permeabilized conditions). Mouse monoclonal antibody U5 (160 mg/ml) followed by FITC goat anti-IgG (dilution 1:40) were used, as described in Materials and Methods. Note the immunostaining in the juxtanuclear area of the aglycosylated PRLR in permeabilized cells (f) and the absence of expression at the plasma membrane level in nonpermeabilized cells (c). 293 cells transfected with WT (g), N80,108D (h), or N35,80,108D (i) PRLR cDNAs were processed for fixation (4% paraformaldehyde) and immunofluorescence staining, as described for COS-7 cells. Note the absence of immunostaining at the cell surface and low perinuclear staining of the aglycosylated receptor in nonpermeabilized conditions (i). Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Transfection, Immunostaining, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 5. Deglycosylated PRLR Colocalizes with the Golgi rab6 in COS-7 Cells a, Labeling of fixed permeabilized COS-7 cells transfected with N35,80,108D receptor using U5 antibody. Panels b and c and panels d and e show a double- labeling experiment of fixed permeabilized COS-7 cells transfected with the N35,80,108D mutant, with anti-PRLR antibody [visualized with a secondary anti-mouse FITC secondary antibody (b and d)], and anti-Golgi antibody [anti-rab6 visualized with bioti- nylated secondary anti-rabbit IgG followed by Texas-Red- conjugated streptavidin (c and e)]. The choice of mouse anti- PRLR and rabbit anti-Golgi IgG as primary antibodies allows accurate double-labeling experiments. Panels b-c and d-e show two different COS-7 cells. f, Micrographs of COS-7 cells stained for anti-protein disulfide isomerase (endoplas- mic reticulum protein) antibody, visualized with Texas-Red- conjugated anti-mouse secondary antibody. Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Labeling, Transfection, Mutagenesis, Staining