Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 2. Heterodimerization of the Cytoplasmic Parts of LEPRb and LEPRa Does Not Induce STAT3 Activation A, COS-7 cells were transfected with expression plasmids encoding the IL-5R and chimeras of LEPRb, LEPRa, gp130, and gp130 without cytoplasmic part (gp130cyt), as indicated. Untransfected cells were supplemented with transfection reagent alone. Cells were cotransfected with 0.3 g of STAT3 expression vector per 6-cm dish. Two days after transfection, cells were stimulated with IL-5 (80 ng/ml) for 30 min before nuclear extracts were prepared. Nuclear extracts were analyzed by Western

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 3. JAK2 Association with gp130, LEPRa, and LEPRb COS-7 cells were cotransfected with expression con- structs encoding the indicated receptor chimeras (/gp130, /LEPRa, /LEPRb, or /gp130box1) and JAK2. After mild lysis of the cells, receptor complexes were immunoprecipi- tated with the anti-IL-5R antibody S-16. Immunocomplexes or cellular lysates were immunoblotted with antibodies against JAK2 or IL-5R as indicated. Observations were con- firmed in four independent experiments.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Expressing, Lysis

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 4. Structure of the Chimeric Receptor Constructs Used in This Study A, Intracellular amino acid sequences of CLRa and CLRb. Presumed box 1 and box 2 motifs (17, 35, 36) are underlined (closed lines); the unique amino acids in the C terminus of CLRa are marked by a dashed line. IL-5R extracellular parts are illustrated as gray boxes; extracellular and transmembrane (TM) regions of the leptin receptor are indicated as white boxes. The positions where stop codons were inserted into CLRb to generate the indicated deletion mutants are illustrated by vertical lines. K889 of LEPR is the last amino acid of CLR-0. B, Schematic representation of deletion mutants of CLRb (in comparison to CLRa and CLRb). The splice site (after amino acid K889) is illustrated by a dashed line. The box 1–3 motifs are represented by black boxes. The membrane proximal 29 amino acids of the cytoplasmic part of the LEPR are identical in LEPRa and LEPRb and include a box 1 motif. Each construct is designated by the number of unique C-terminal amino acids. The amino acids unique in CLRa are illustrated by a hatched box. The deletion constructs contain no intracellular tyrosine residue. Lengths of receptor constructs are not drawn to scale.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Construct, Comparison, Membrane, Residue

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 5. Signaling of Deletion Mutants of CLRb in Heterodimeric Complexes with CLRb A and B, COS-7 cells were cotransfected with expression plasmids encoding the indicated chimeric receptor constructs and a STAT3 expression vector. Thirty-two hours after transfection, cells were starved for 16 h and then treated with 80 ng/ml IL-5 or vehicle only for 30 min before nuclear extracts were prepared. Extracts were used for Western blot analysis with a phospho-specific STAT3 antibody. For expression control, lysates were probed with antibodies against STAT3, human (h)IL-5R, and hIL-5R. C, HepG2 cells were transfected with expression plasmids encoding /CLRb and the indicated chimera along with an 2-macroglobulin promoter luciferase reporter gene construct. One day after transfection, cells were stimulated with IL-5 (80 ng/ml) or vehicle only for 24 h. Luciferase activity of lysates was normalized to the activity of coexpressed -galacto- sidase. One representative experiment is shown. Columns represent the average of two independent transfections and bars reflect the differences between the two. The same differences between the average values of the expression constructs were observed in three additional experiments.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Expressing, Construct, Plasmid Preparation, Transfection, Western Blot, Control, Luciferase, Activity Assay

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 7. Mutational Analysis of a 13-Amino Acid Region Crucial for STAT3 Signaling by CLRb A, Design of point mutants from CLR-15 and CLR-19. Sequences of the membrane proximal cytoplasmic segments of the LEPR of Mus musculus (EMBL database accession no. Y10298; upper sequence) and Gallus gallus (AF169827; lower sequence) are aligned. In CLR-15 and CLR-19, conserved amino acids were replaced by alanine (arrows). The box 1 motif (solid lines) and the proposed box 2 motif (dashed lines) (17) are boxed. The transmembrane region (TM) is indicated. B, COS-7 cells were transfected with expression plasmids encoding the IL-5R and chimeras as indicated and a STAT3 expression vector. The experiment was performed as described in Fig. 5. Results for each construct were confirmed in three independent experiments.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Membrane, Sequencing, Transfection, Expressing, Plasmid Preparation, Construct

Journal: PLoS ONE

Article Title: T-Helper Type-2 Contact Hypersensitivity of Balb/c Mice Aggravated by Dibutyl Phthalate via Long-Term Dermal Exposure

doi: 10.1371/journal.pone.0087887

Figure Lengend Snippet: Representative images of the expression of (A) IL-5 and (B) IL-13 as determined by immunohistochemical staining (brown color stain) (black arrow). Panel: (A1, B1) Saline, (A2, B2) DBP 0.4, (A3, B3) DBP 4, (A4, B4) DBP 40, (A5, B5) FITC, (A6, B6) FITC+DBP 0.4, (A7, B7) FITC+DBP 4, (A8, B8) FITC+DBP 40. Analyses of (C) IL-5 and (D) IL-13 expression levels according to average optical density. Animal groups (in all panels): n = 4 mice per group. **: p <0.01, compared with saline group; ##: p <0.01, compared with FITC group.

Article Snippet: Mouse anti-IL-5-antibody, mouse anti-IL-13-antibody, and mouse anti-eosinophil cationic protein (ECP)-antibody, goat-anti-rabbit lgG-antibody, a rabbit lgG peroxidase conjugated streptavidin-biotin complex (SABC-POD) kit and a diaminobenzidine (DAB) kit were obtained from Boster Bio-engineering (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Staining