Journal: The Journal of clinical endocrinology and metabolism

Article Title: GPIHBP1 C89F neomutation and hydrophobic C-terminal domain G175R mutation in two pedigrees with severe hyperchylomicronemia.

doi: 10.1210/jc.2011-1444

Figure Lengend Snippet: FIG. 2. Binding of human LPL to GPIHBP1 variants at the cell surface. A, Western blot analysis. CHO pgsA-745 cells were transiently cotransfected with a human Myc-Tagged LPL vector and an empty vector, an expression vector for WT human GPIHBP1 (WT), GPIHBP1-C14F, GPIHBP1-C89F, GPIHBP1-C14F/C89F, or GPIHBP1-G175R. The amount of LPL bound to the cells was determined after incubation of cells with a rabbit antibody against Myc tag, revealed in Western blot by a HRP-conjugated goat antirabbit IgG. The levels of total GPIHBP1 and LPL expression were assessed with an antibody against the Flag-tag. Actin was used as control. The results are a representative series of three independent experiments. B and C, Quantification of Western blots; B, amounts of LPL bound to GPIHBP1 at the cell surface; C, amounts of LPL bound to GPIHBP1 normalized on total LPL in cells. All signals was normalized to actin signals and expressed relative to the ratio for WT GPIHBP1 (set at 100%). Data are expressed as mean SEM of three independent experiments. *, P 0.05 compared with WT.

Article Snippet: Western blots were performed with a HRP-conjugated goat antirabbit IgG (1:1000; Bio-Rad).

Techniques: Binding Assay, Western Blot, Plasmid Preparation, Expressing, Incubation, FLAG-tag, Control

Journal: RNA

Article Title: Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns

doi: 10.1261/rna.048520.114

Figure Lengend Snippet: Comparison of zebrafish CTE function in 293T cells with added Nxf1 and Nxt from zebrafish and mammals. ( A ) 293T cells (1.2 × 10 6 ) were transfected with 1.8 µg of the CTE-reporter plasmid pCMVGagPol-DrCTE(2X) (HR4466), 90 ng of pCMV-SEAP (HR1931), and plasmids expressing zebrafish or mammalian Nxf1 and Nxt proteins as indicated in the figure. The HsNxf1, DrNxf1, and DrNxt2 proteins contain an amino-terminal Flag-epitope tag. The MmNxt1 protein is not Flag-tagged. DNA transfections, p24 and SEAP activity analyses were performed as described in the legend of A. ( B ) Western blot analysis of protein expression in the transfected 293T cells. Lysates from the transfected cells were separated on denaturing SDS-PAGE gels and analyzed by Western blotting. Blots were probed with an M2 monoclonal anti-Flag antibody (Sigma) and detected with a goat anti-mouse IgG antibody-IRDye 800. Blots were visualized and analyzed using the LI-COR Odyssey infrared imaging system.

Article Snippet: For detection of Flag-tagged proteins, the blots were probed with a 1:5000 dilution of M2 anti-Flag monoclonal antibody (Sigma), followed by incubation with a goat anti-mouse IgG antibody-IRDye 800 (1:25,000 dilution; LI-COR).

Techniques: Transfection, Plasmid Preparation, Expressing, FLAG-tag, Activity Assay, Western Blot, SDS Page, Imaging

Journal: PLoS ONE

Article Title: Antibodies Elicited in Response to EBNA-1 May Cross-React with dsDNA

doi: 10.1371/journal.pone.0014488

Figure Lengend Snippet: (A) Anti-EBNA-1 ELISA. 3D4 was tested by ELISA, at increasing concentrations, for binding to EBNA-1, BSA, and the cystovirus, polymerase protein, P2 (negative control) coated on Costar plates. Plates were read at 405 nm at 20 minutes. 3D4 shows specificity for EBNA-1 under these conditions. Results are the averages of triplicates and standard deviations are indicated. (B) 3D4 was tested by ELISA for binding to dsDNA coated on Immulon-2 plates. Plates were read at 405 nm at 1 hour. Results in A and B are the average of triplicates and standard deviations are indicated. (C) 3D4 antibody is of the IgG1 isotype. ELISA plates coated with unlabeled anti-IgG1, anti-IgG2a, anti-IgG2b, or anti-IgG3 were incubated with 1.5 µg/ml of 3D4 MAb followed by the respective polyclonal isotype specific antibodies conjugated to alkaline phosphatase. (D) Purified 3D4 at a concentration of 10 µg/ml was examined for binding to Crithidia luciliae slides. Left panel shows the binding of 3D4 to kinetoplasts of Crithidia luciliae (arrow). Right panel; IgG1 isotype control antibody, does not bind specifically to kinetoplasts. (E & F) 3D4 was adsorbed on a dsDNA cellulose column and pre and post adsorbed antibody were tested for binding to dsDNA and EBNA-1 by ELISA (E) and to EBNA-1 by Western blot (F). (E) 3D4 adsorbed on dsDNA cellulose beads was completely depleted of antibody with reactivity for dsDNA and EBNA-1 as detected by ELISA. Results represent OD 405 nm values after subtraction of non specific binding to cellulose only beads. Standard deviations of triplicate wells are indicated. Anti-dsDNA and anti-EBNA-1 ELISAs were performed on Immulon-2 and Costar plates respectively and ELISAs were developed when ODs on each plate reached maximal values. (F) Post dsDNA cellulose adsorbed 3D4 shows reduced binding to EBNA-1 by Western blot. Left panel: Coomassie stained polyacrylamide gel. Right panel: Western blot: filters were immunostained with pre (lanes 2 and 3) or post dsDNA cellulose adsorbed 3D4 MAb (lane 4). Molecular weight markers used in Western blot were conjugated to strep-tag and were detected with Strep-Tactin-HRP.

Article Snippet: ELISA plates were coated with 50 μl of a 1∶1000 dilution of either unlabeled goat anti-mouse IgG1, IgG2a, IgG2b or IgG3 (Southern Biotech, Birmingham, Alabama) and incubated at 37°C for one hour and overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Incubation, Purification, Concentration Assay, Control, Western Blot, Staining, Molecular Weight, Strep-tag

Journal: Cell Metabolism

Article Title: Estradiol regulates leptin sensitivity to control feeding via hypothalamic Cited1

doi: 10.1016/j.cmet.2023.02.004

Figure Lengend Snippet:

Article Snippet: Goat anti-Iba1 , Novus/Biotechne , #NB100-1028; RRID: AB_521594.

Techniques: Virus, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Diagnostic Assay, Expressing, Software