Journal: Vaccines

Article Title: Mimicking Native Display of CD0873 on Liposomes Augments Its Potency as an Oral Vaccine against Clostridioides difficile

doi: 10.3390/vaccines9121453

Figure Lengend Snippet: Immune responses in hamsters immunised orally with CD0873 alone and CD0873-MalLipo. Intestinal fluid and serum were harvested from vaccinated hamsters and from negative control hamsters and tested for the presence of mucosal IgA and serum IgG, respectively. Intestinal fluid was extracted by flushing the small intestine with 5 mL of PBS containing protease inhibitors, and serum was taken by cardiac puncture. ( A ) Intestinal fluid (5 µL) from each hamster was first fractionated by SDS-PAGE and transferred to a PVDF membrane then probed with rabbit anti-hamster IgA antibody (1:1000). Bands were detected with anti-rabbit IgG HRP (1:1000) and TMB substrate, and band intensity was quantified using ImageJ. ( B ) Serum diluted 1:10 of CD0873- and CD0873-MalLipo-vaccinated group and naïve group tested for IgG by indirect ELISA. Goat anti-hamster IgG highly cross-adsorbed Biotin antibody (1:20,000) and Streptavidin-HRP (1:200) were used for detection. Data were obtained from two independent experiments, each with three technical replicates. Error bars represent standard error of the mean (SEM). All data were analysed by Kruskal–Wallis test followed by Dunn’s multiple comparison. Error bars represent the standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Wells were washed, then incubated for 2 h at room temperature in 100 µL goat anti-hamster IgG highly cross-adsorbed Biotin antibody (Sigma Merck Group, Feltam, UK) diluted 1:20,000 in PBST, washed again, then incubated for 2 h in Streptavidin-HRP (R&D Systems, Minneapolis, MN, USA) diluted 1:200 in PBST.

Techniques: Negative Control, SDS Page, Indirect ELISA

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tetraspanin CD9 negatively regulates lipopolysaccharide-induced macrophage activation and lung inflammation.

doi: 10.4049/jimmunol.0802797

Figure Lengend Snippet: FIGURE 4. The expression of CD14, complex formation of CD14 and TLR4, and their localization into the lipid raft are enhanced in CD9 KO BMDMs. A, BMDMs from WT and CD9 KO mice were stimulated with 1 g/ml LPS. After the indicated hours, cell lysates were electrophoresed on SDS-PAGE, transferred to a membrane, probed with anti-CD14 and anti-TLR4 Abs. Anti-actin blots show comparable amounts of protein loaded in each lane. B, WT and CD9 KO BMDMs were cultured in the absence () or presence () of LPS for 20 h. CD14 protein in whole cell lysates (WCL) and in immuno- precipitates with anti-TLR4 mAb (MTS510) was immunoblotted with biotinylated anti-CD14 Ab after SDS-PAGE. Anti-actin blots show comparable amounts of protein loaded in each lane. C, Cell lysates from WT and CD9 KO BMDMs cultured in the absence () or presence () of LPS for 2 h were centrifuged in sucrose density gradients. Fractions were collected from the top of the gradient and separated by SDS-PAGE. Protein distribution in the fractions was visualized by immunoblotting with anti-CD14, anti-TLR4, anti-CD9, anti-CD81, anti-CD45, and anti-flotillin-1 Abs. GM1 ganglioside was detected with HRP-conjugated cholera toxin by dot blot. The intensity of blots was quantified by densitometry. Percentage of density units of light membrane (LM) fractions (4 plus 5) were calculated and shown on the right of blots. D, Pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10) from the sucrose gradients of WT cell lysate were subjected to imunoprecipiation with anti-CD9 mAb. After separation of the immunoprecipitates on SDS-PAGE, CD14 and CD9 were immunoblotted with biotinylated anti-CD14 Ab and anti-CD9 mAb, respectively. Data shown are from one representative of three similar experiments.

Article Snippet: Rat anti-CD14 mAb (rmC5–3) and goat biotinylated anti-CD14 polyclonal Ab (BAF9892) were purchased from BD Biosciences and R&D Systems, respectively.

Techniques: Expressing, SDS Page, Membrane, Cell Culture, Western Blot, Dot Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tetraspanin CD9 negatively regulates lipopolysaccharide-induced macrophage activation and lung inflammation.

doi: 10.4049/jimmunol.0802797

Figure Lengend Snippet: FIGURE 5. Accelerated IB degradation and analysis of other signaling molecules in LPS-activated CD9 KO BMDMs. A, BMDMs from WT and CD9 KO mice were stimulated with 1 g/ml LPS. After the indicated minutes, cell lysates were separated by SDS-PAGE, transferred to a mem- brane, and immunoblotted with anti-IB, anti-phosphorylated p38 (p- p38), and anti-p38 polyclonal Abs. Anti-actin blots show comparable amounts of protein loaded in each lane. B, SHP-1 was immunoprecipitated using anti-SHP-1 polyclonal Ab from cell lysates of WT and CD9 KO BMDMs stimulated with LPS for the indicated hours. After electrophoresis on SDS-PAGE and transfer to a membrane, tyrosine-phosphorylated SHP-1 was probed with anti-phosphotyrosine mAb (PY20), and the asso- ciated SIRP was blotted with anti-SIRP polyclonal Ab. SHP-1 and SIPR in whole cell lysates (WCL) were immunoblotted in parallel. Anti- actin blots show comparable amounts of protein loaded in each lane. Data shown are from one representative of three similar experiments.

Article Snippet: Rat anti-CD14 mAb (rmC5–3) and goat biotinylated anti-CD14 polyclonal Ab (BAF9892) were purchased from BD Biosciences and R&D Systems, respectively.

Techniques: SDS Page, Immunoprecipitation, Electrophoresis, Membrane