Journal:

Article Title: Rotavirus Infection Reduces Sucrase-Isomaltase Expression in Human Intestinal Epithelial Cells by Perturbing Protein Targeting and Organization of Microvillar Cytoskeleton

doi:

Figure Lengend Snippet: DPP IV biosynthesis, maturation, and stability in RRV-infected cells. After the immunoprecipitation with an anti-SI MAb (Fig. ​(Fig.3),3), the supernatants depleted of SI were immunoprecipitated with an anti-DPP IV MAb. The immunoprecipitates were then analyzed by SDS-PAGE on a 6 to 15% (A) or 7.5% (B) polyacrylamide gel without endo H treatment (panel B, lanes 1, 3, 5, 7, 9, and 11) or after endo H treatment (panel B, lanes 2, 4, 6, 8, 10, and 12) in order to differentiate the high-mannose DPP IV form from viral protein VP4 as described in Materials and Methods. After endo H treatment, the immature form of DPP IV became deglycosylated and its molecular mass decreased to 85 kDa, whereas the molecular mass of VP4 did not change. Labeled proteins were visualized by fluorography (black square, immature 100-kDa DPP IV; white square, deglycosylated 85-kDa DPP IV; black arrowhead, mature 110-kDa DPP IV; white arrowhead, viral protein VP4). I, RRV-infected Caco-2 cells; C, mock-infected Caco-2 cells.

Article Snippet: Endoglycosidase H (endo H) was provided by Genzyme (Tebu, Le Perray en Yveline, France).

Techniques: Infection, Immunoprecipitation, SDS Page, Labeling

Journal:

Article Title: Intracellular Complexes of Viral Spike and Cellular Receptor Accumulate during Cytopathic Murine Coronavirus Infections

doi:

Figure Lengend Snippet: Coimmunoprecipitation of MHVR and S from cells producing both proteins. (A) Rhi or Rlo cells were coinfected with vaccinia virus recombinants vTF7.3 and vTM1-SJHM and then radiolabeled with Tran[35S]-label from 3 to 7 h postinfection. Immediately after labeling, cytoplasmic extracts were prepared by the NP-40 lysis procedure, and 35S-labeled proteins were immunoprecipitated with antireceptor antiserum and Sepharose-protein G. Proteins bound to the Sepharose-protein G were treated with endoglycosidase H (+) or left untreated (−) and then prepared for SDS-polyacrylamide gel electrophoresis. The separated proteins were visualized by fluorography. Lane 1, Rhi cells (vTF7.3 plus VVWR) (control; no S produced); lanes 2 and 3, Rhi cells (vTF7.3 plus vTM1-SJHM); lane 4, Rlo cells (vTF7.3 plus vTM1-SJHM); lanes 5 and 6, Rhi cells (vTF7.3 plus vTM1-Secto. (B) Rhi cells were coinfected with vTF7.3 and vTM1-SJHM. At 12 h postinfection, cells were lysed along with exogenously added 35S-labeled A59 virions (12 × 104 cpm) and then immunoprecipitated with antireceptor antibody as described above. Precipitations were performed in the absence or presence of exogenously added sMHVR, which specifically absorbs S proteins to Sepharose-protein G (28). Immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and fluorography. Lane 7, vTF7.3 plus VVWR; lanes 8 and 9, vTF7.3 plus vTM1-SJHM.

Article Snippet: Samples in the endoglycosidase H buffer were incubated for 4 h at 37°C in the presence of 0.5 mU of endoglycosidase H (Boehringer Mannheim).

Techniques: Labeling, Lysis, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Produced

Journal: bioRxiv

Article Title: 13 C ENDOR Spectroscopy-Guided MD Computations Reveals the Structure of the Enzyme-Substrate Complex of an Active, N-linked Glycosylated Lipoxygenase

doi: 10.1101/2022.12.07.519351

Figure Lengend Snippet: 35 GHz 13 C Mims ENDOR of native ( black ) and EndoH ( red ) Mo LOX with 13 C10-LA, 13 C11-LA. ENDOR contributions from two electron-spin transitions as mentioned. Conditions same as in . H = 10.9 kG. The complete 2D ENDOR spectra are in Figure S6 .

Article Snippet: An endoglycosidase H (EndoH) gene from Streptomyces plicatus was synthesized (GenScript) for recombinant expression in E. coli and subcloned in-line with his-tagged MBP construct (2CT-10 vector, Addgene plasmid #55209).

Techniques: