Journal: Journal of Biological Chemistry

Article Title: Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

doi: 10.1074/jbc.m705725200

Figure Lengend Snippet: FIGURE 2. IL-15 stabilizes IL-15R. Human 293 cells were transfected with plasmids expressing 0.05 g IL-15R (A) or IL-15sR (B) alone or in combina- tion with 0.05 g of IL-15t, as indicated. After 72 h, IL-15R production in the culture supernatants and cell extracts was analyzed by Western immunoblot using a goat anti-IL-15R antibody. Sample dilutions of 1:2 and 1:4 were loaded as indicated to quantify the produced IL-15R. Mock indicates trans- fectionwithacontrolplasmidonly(GFP).Theamountofundilutedcellextract and culture supernatants loaded on the gel were 1:200 and 1:300, respec- tively. Arrows indicate the position of IL-15R bands, and arrowheads indicate the position of molecular weight markers. Similar transfection efficiencies were verified by co-transfection of GFP expression vectors. C, mRNA expres- sion levels of IL-15 and IL-15R plasmids transfected individually or together in human 293 cells. The expression of cellular gene GAPDH was used as con- trol (lower panel). The location of mRNAs was identified by Northern blot analysis and is indicated by arrows.

Article Snippet: Human IL-15 levels were measured by ELISA (Quantikine human IL-15 immunoassay; R & D Systems) or by Western immunoblot (using the polyclonal goat anti-human IL-15 antibody AF315, R & D Systems).

Techniques: Transfection, Expressing, Western Blot, Produced, Molecular Weight, Cotransfection, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

doi: 10.1074/jbc.m705725200

Figure Lengend Snippet: FIGURE4.IntracellularassociationofIL-15withtheIL-15R.A,human293 cells were transfected with IL-15-expressing plasmid (IL-15t) alone or in com- bination with IL-15R-expressing plasmid producing either full-length (IL- 15R) or soluble form (IL-15sR). Complexes were immunoprecipitated from cell extracts or culture medium with an anti-IL-15R antibody and were sub- sequently examined by Western immunoblot analysis using an anti-IL-15 antibody. The three bands corresponding to the unglycosylated, partially, and fully N-glycosylated forms of IL-15 are indicated. B, human 293 cells were transfected with 0.05 g of IL-15FLAG DNA alone, IL-15R DNA alone, or IL-15FLAGIL-15RDNAsincombination.Cellstransfectedwiththeindivid- ual plasmids were harvested and washed separately at 4 °C and divided into two tubes. Recombinant human IL-15 protein was added to the cells as indi- cated using 50 excess of the expected IL-15 production. The cells were lysed, and IL-15 was immunoprecipitated using anti-FLAG antibody. The complexes were separated on SDS-PAGE, and the IL-15R was visualized by Western immunoblot. The positions of the glycosylated and unglycosylated IL-15R are indicated.

Article Snippet: Human IL-15 levels were measured by ELISA (Quantikine human IL-15 immunoassay; R & D Systems) or by Western immunoblot (using the polyclonal goat anti-human IL-15 antibody AF315, R & D Systems).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Recombinant, SDS Page

Journal: Protein engineering, design & selection : PEDS

Article Title: Evolution of interleukin-15 for higher E. coli expression and solubility.

doi: 10.1093/protein/gzq107

Figure Lengend Snippet: Fig. 2 Results of second-step screening process of IL-15-CAT fusions when expressed in E. coli DH5a. The vertical axis (solubility test) corresponds to the ELISA absorbance of cell extracts incubated in plates coated with Mab247, revealed with an anti-CAT polyclonal antibody. The horizontal axis (functionality test) corresponds to the ELISA absorbance of cell extracts incubated in plates coated with IL-15Ra and revealed with Mab247. Filled squares represent clones selected at cam ,400 mM; filled circle, clones selected at cam 800 mM; filled triangle, clones selected at cam 1200 mM; open square, clones selected at cam 1500 mM.

Article Snippet: To screen mutants for their solubility, a sandwich ELISA test was used with Mab247 (anti-human IL-15 IgG, R&D system), dilution 1 mg/ml, as the capture antibody and a two-step detection system with a polyclonal rabbit anti-CAT IgG (Sigma) and a goat anti-rabbit IgG conjugated with peroxidase (Sigma).

Techniques: Solubility, Enzyme-linked Immunosorbent Assay, Incubation, Clone Assay