Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 4. Perinuclear Localization of the Aglycosylated PRLR COS-7 cells transfected with WT (a and d), N80,108D (b and e), or N35,80,108D (c and f) PRLR cDNAs were fixed in 4% paraformaldehyde (a, b, and c: nonpermeabilized conditions) or in methanol, 220 C (d, e, and f: permeabilized conditions). Mouse monoclonal antibody U5 (160 mg/ml) followed by FITC goat anti-IgG (dilution 1:40) were used, as described in Materials and Methods. Note the immunostaining in the juxtanuclear area of the aglycosylated PRLR in permeabilized cells (f) and the absence of expression at the plasma membrane level in nonpermeabilized cells (c). 293 cells transfected with WT (g), N80,108D (h), or N35,80,108D (i) PRLR cDNAs were processed for fixation (4% paraformaldehyde) and immunofluorescence staining, as described for COS-7 cells. Note the absence of immunostaining at the cell surface and low perinuclear staining of the aglycosylated receptor in nonpermeabilized conditions (i). Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Transfection, Immunostaining, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 5. Deglycosylated PRLR Colocalizes with the Golgi rab6 in COS-7 Cells a, Labeling of fixed permeabilized COS-7 cells transfected with N35,80,108D receptor using U5 antibody. Panels b and c and panels d and e show a double- labeling experiment of fixed permeabilized COS-7 cells transfected with the N35,80,108D mutant, with anti-PRLR antibody [visualized with a secondary anti-mouse FITC secondary antibody (b and d)], and anti-Golgi antibody [anti-rab6 visualized with bioti- nylated secondary anti-rabbit IgG followed by Texas-Red- conjugated streptavidin (c and e)]. The choice of mouse anti- PRLR and rabbit anti-Golgi IgG as primary antibodies allows accurate double-labeling experiments. Panels b-c and d-e show two different COS-7 cells. f, Micrographs of COS-7 cells stained for anti-protein disulfide isomerase (endoplas- mic reticulum protein) antibody, visualized with Texas-Red- conjugated anti-mouse secondary antibody. Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Labeling, Transfection, Mutagenesis, Staining

Journal: Cell Reports

Article Title: NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

doi: 10.1016/j.celrep.2013.06.029

Figure Lengend Snippet: Processing of VTRNA1.1 into svRNAs, Related to Figure 4 (A) Complete list of svRNAs derived from VtRNAs found in human fibroblasts. (B) QPCR showing expression of NSun2 RNA relative to GAPDH in NSUN2 (−/−) and (+/−) fibroblasts. (C) Abundance of svRNA4 in NSUN2 −/− cell lysates (no-RNA) or incubated with synthetic VTRNA1.1 carrying (m 5 C) or lacking (no-m 5 C) at position 69 at 37°C. (D) Western Blot detecting CACNG8 protein (43kDa) (upper panel) in NSUN2 (−/−) and (+/−) fibroblasts in two independent replicates (R1, R2). Bands at higher molecular weight are unspecific. Actin served as a loading control (lower panel). (E) Western Blot detecting NSun2 (100kDa) (upper panel) and CACNG8 protein (43kDa) (middle panel) in NSUN2 −/− cells infected with an empty vector control (eV) or full length NSun2 (NSun2). Tubulin (50kDa) (lower panel) served as loading control.

Article Snippet: TaqMan probe sets used were CACNG7: Hs00259061_m1, CACNG8: Hs01100182_m1, NSUN2: Hs00214829_m1, and GAPDH (4326317E) as an internal control.

Techniques: Derivative Assay, Expressing, Incubation, Western Blot, Molecular Weight, Control, Infection, Plasmid Preparation

Journal: Cell Reports

Article Title: NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

doi: 10.1016/j.celrep.2013.06.029

Figure Lengend Snippet: Differential Processing of vtRNA1.1 into svRNAs in the Absence of m 5 C (A) Schematics of secondary structure of vtRNA1.1 and small vault RNA (svRNA) found to be differentially abundant in NSUN2 +/− and NSUN2 −/− fibroblasts. CH3, cytosine-5 methylated site at position 69. (B) Fold-change (log 2 ) and false discovery rate (FDR) values for reads of svRNA1-4 in NSUN2 +/− versus NSUN2 −/− human fibroblasts. (C) Detection of svRNA1 and svRNA4 in NSUN2 +/− and NSUN2 −/− cells using qPCR. (D and E) RNA levels of NSun2 (D) and svRNA4 (E) in NSun2 null (−/−) fibroblasts rescued by viral infection of NSun2 (pB-NSun2) compared to the empty vector control (pB-empty). (F) Abundance of svRNA4 in NSUN2 −/− cell lysates (no-RNA) or incubated with synthetic vtRNA1.1 carrying (m 5 C) or lacking (no-m 5 C) at position 69. (G) Detection of svRNA4 in small RNA pool copurified with Argonaute 2 (left) and Argonaute 3 (right). Let7-a and mir-150 are negative and mir-21 and mir-92-b are positive controls for Argonaute 2- and 3-bound microRNAs, respectively. (H) qPCR showing expression of CACNG7 and CACNG8 RNA relative to GAPDH in NSUN2 −/− and NSUN2 +/− fibroblasts. (I) Fold-change expression of CACNG7 and CACNG8 RNA in NSUN2 −/− and NSUN2 +/− fibroblasts transduced with svRNA antagomirs (as-svRNA4) or svRNA4 microRNA mimics (svRNA) versus respective control RNAs (ctr-RNA). (J) RNA levels of CACNG7 and CACNG8 relative to GAPDH in NSun2 null (−/−) fibroblasts rescued by viral infection of NSun2 (pB-NSun2) compared to the empty vector control (pB-empty). Error estimates represent SEM (C–J). See also and .

Article Snippet: TaqMan probe sets used were CACNG7: Hs00259061_m1, CACNG8: Hs01100182_m1, NSUN2: Hs00214829_m1, and GAPDH (4326317E) as an internal control.

Techniques: Methylation, Infection, Plasmid Preparation, Control, Incubation, Expressing, Transduction