Journal:

Article Title: Upregulation of CD40 Expression on Endothelial Cells Infected with Human Cytomegalovirus

doi: 10.1128/JVI.76.24.12803-12812.2002

Figure Lengend Snippet: Immunoblot analysis of CD40 in whole-cell lysates. Cell monolayers were infected with HCMV TB40E at an MOI of 1 (inf.) or mock infected (noninfected [n.i.]). Cell lysates were prepared at the indicated times and analyzed by immunoblotting. (A) CD40 protein levels were detected with a polyclonal rabbit serum directed against human CD40. (B) Blots from panel A were stripped and reprobed with a rabbit serum directed against calreticulin. (C) Signal intensities from panels A and B were densitometrically quantified; data represent the ratio of CD40 to calreticulin (y axis) versus time p.i. (x axis). (D) Protein lysates from infected or noninfected cells were treated with N-glycosidase F (+) or left untreated (−) prior to immunoblot analysis with anti-CD40 antibodies. Molecular masses are given in kilodaltons.

Article Snippet: The following monoclonal antibodies or polyclonal sera were used: mouse IgG1-fluorescein isothiocyanate (FITC)-conjugated anti-human CD54 (Calbiochem, La Jolla, Calif.), anti-human major histocompatibility complex class I (MHC-I) (BD PharMingen, San Diego, Calif.), mouse IgG1-FITC anti-human CD40 (Dianova, Hamburg, Germany), mouse IgG1-FITC anti-human CD62E (Calbiochem), antibodies G28.5 and Ro1 (anti-human CD40) (gifts from H. Engelmann, Institute for Immunology, University of Munich, Munich, Germany) ( 39 ), polyclonal rabbit anti-CD40 serum (Santa Cruz Biotechnology), rabbit antiserum against human calreticulin (Dianova), biotin-conjugated rabbit anti-mouse antibodies, and biotinylated swine anti-rabbit antibodies (Dako, Hamburg, Germany).

Techniques: Western Blot, Infection

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium

doi: 10.1186/1477-7827-9-56

Figure Lengend Snippet: PMP22 is expressed in the endometrium . (A). Frozen adult human tissues were obtained, and total RNA isolated. PMP22 exon 1B mRNA was expressed in endometrium, lung, and tonsil tissue while expression of PMP22 exon 1B mRNA was only observed in the lung. GAPDH mRNA was used as the loading control. (B) Tissue lysates were prepared to determine PMP22 protein expression in the endometrium. PMP22 protein was detectable in proliferative endometrium, and a representative sample is depicted. N = 6. (C) PMP22 expression was confirmed in human proliferative endometrium using immunohistochemistry, and a representative sample is displayed. N = 6. Magnification = 400 ×. (D) PMP22 mRNA transcripts were detected in HEC-1A, Ishikawa, and RL95-2 endometrial cell lines using semi-quantitative RT-PCR. GAPDH was used as a loading control. (E) PMP22 protein levels in the three immortalized endometrial cell lines were detected using PMP22 antisera. β-actin was used as a loading control. (E) To study PMP22 in HEC-1A epithelial cells, its expression was modulated through ectopic overexpression, or by inhibition using a PMP22 specific siRNA. Vector and scrambled siRNA controls are included for endogenous expression. Expression of PMP22 was confirmed by western blot analysis, with β-actin levels serving as a loading control.

Article Snippet: Pre-cleared lysates were incubated overnight with Protein-A beads bound to either anti-PMP22 rabbit polyclonal antisera (Sigma-Aldrich), anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology), or control rabbit sera.

Techniques: Isolation, Expressing, Immunohistochemistry, Quantitative RT-PCR, Over Expression, Inhibition, Plasmid Preparation, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium

doi: 10.1186/1477-7827-9-56

Figure Lengend Snippet: PMP22 expression differentially regulates select integrin pairs . The surface expression of α6, αvβ3, or α2 integrin in HEC-1A/PMP22, HEC-1A/Vector, or HEC-1A/PMP22 siRNA cells was determined using flow cytometry. The MFI was quantitated and tabulated in the top corner of each histogram. Upregulation of PMP22 promoted the surface expression of α6 integrin, while slightly reducing αvβ3 integrin. Reciprocal effects were seen with the PMP22 specific siRNA. In contrast, no change in α2 integrin expression was observed when PMP22 expression was modulated. The experiment was repeated three times, and a representative experiment is shown. Statistical differences between α6 integrin (B) and αvβ3 (C) expression were determined between the three cell lines. *, p < 0.05; Student's unpaired t test.

Article Snippet: Pre-cleared lysates were incubated overnight with Protein-A beads bound to either anti-PMP22 rabbit polyclonal antisera (Sigma-Aldrich), anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology), or control rabbit sera.

Techniques: Expressing, Plasmid Preparation, Flow Cytometry

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium

doi: 10.1186/1477-7827-9-56

Figure Lengend Snippet: PMP22 expression increased binding on laminin . (A) 5 × 10 4 cells (HEC-1A/PMP22, HEC-1A/Vector, HEC-1A/scrambled siRNA control, and HEC-1A/PMP22 siRNA) were incubated in serum-free media at 37°C for 30 min in wells coated with different extracellular matrices. Absorbance was measured at 595 nm. Values are represented as the mean ± SEM. for triplicate wells (* p < 0.05; Student's unpaired t test). A representative experiment is shown; three independent experiments yielded similar results. (B) In order to confirm that laminin binding was α6 integrin dependent, HEC-1A/PMP22 cells were incubated with indicated concentrations of α6 or α2 (control) integrin antibodies. Samples were then plated on laminin for 30 min, and analyzed as above.

Article Snippet: Pre-cleared lysates were incubated overnight with Protein-A beads bound to either anti-PMP22 rabbit polyclonal antisera (Sigma-Aldrich), anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology), or control rabbit sera.

Techniques: Expressing, Binding Assay, Plasmid Preparation, Incubation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium

doi: 10.1186/1477-7827-9-56

Figure Lengend Snippet: PMP22 and α6 integrin associate with each other . HEC-1A/Vector or HEC-1A/PMP22 cells were lysed in 1% NP-40 and incubated with PMP22 or α6 integrin antisera. Immunoprecipations were assessed by SDS-PAGE analysis. (A) α6 integrin and PMP22 co-immunoprecipitate in HEC-1A/Vector and HEC-1A/PMP22 cells when using either PMP22 or the α6 integrin antibody. Normal rabbit sera were used to assess nonspecific pull-down of proteins. (B) Confocal images of HEC-1A/PMP22 cells after co-immunolabeleing with anti-PMP22 (Rhodamine) and anti-α6 integrin (clone G0H3; FITC) antibodies. Co-localization of PMP22 protein (Rhodamine) with α6 integrin (FITC) was observed in both intracellular and plasma membrane compartments. Scale bar, 10 μM. Insets, 50 μM.

Article Snippet: Pre-cleared lysates were incubated overnight with Protein-A beads bound to either anti-PMP22 rabbit polyclonal antisera (Sigma-Aldrich), anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology), or control rabbit sera.

Techniques: Plasmid Preparation, Incubation, SDS Page

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium

doi: 10.1186/1477-7827-9-56

Figure Lengend Snippet: PMP22 mRNA levels inversely correlate in proliferative and secretory phase samples . Normal proliferative (N = 6) and secretory (N = 6) tissue was analyzed for PMP22 expression levels. GAPDH was used to normalize between samples. Significant differences exist in PMP22 expression between normal proliferative and secretory tissues. * p = 0.02.

Article Snippet: Pre-cleared lysates were incubated overnight with Protein-A beads bound to either anti-PMP22 rabbit polyclonal antisera (Sigma-Aldrich), anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology), or control rabbit sera.

Techniques: Expressing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium

doi: 10.1186/1477-7827-9-56

Figure Lengend Snippet: PMP22 and α6 integrin share a similar pattern of expression in human endometrium . (A, C) Normal human proliferative endometrium was stained by immunohistochemistry for either PMP22 or α6 integrin, respectively. Bound antibodies were visualized using DAB, and nuclei counterstained using hemotoxylin. Magnification: 100X. Insets depict a 400X enlargement of glandular epithelium. (B, D) Normal human secretory endometrium was stained by immunohistochemistry for either PMP22 or α6 integrin as above. Magnification: 100X. Insets depict a 400X enlargement of glandular epithelium. (E) Normal human proliferative endometrium was stained as above using non-specific control rabbit sera. (F) The mean integrated intensity of PMP22 and α6 integrin protein expression was quantitated for each sample; N = 6 per group. The error bars represent the standard error of the mean. * p < 0.05.

Article Snippet: Pre-cleared lysates were incubated overnight with Protein-A beads bound to either anti-PMP22 rabbit polyclonal antisera (Sigma-Aldrich), anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology), or control rabbit sera.

Techniques: Expressing, Staining, Immunohistochemistry