Journal: Journal of Biological Chemistry

Article Title: JPDI, a Novel Endoplasmic Reticulum-resident Protein Containing Both a BiP-interacting J-domain and Thioredoxin-like Motifs

doi: 10.1074/jbc.m208346200

Figure Lengend Snippet: FIG. 3. Subcellular localization of JPDI-HA. A, NIH3T3 cells were transiently transfected with pCAG-JPDI-HA and incubated for 36 h. Cells then were fixed and co-stained with mouse 12CA5 anti-HA and rabbit anti-PDI antibodies. Fluorescence microscopic images were obtained using fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies. B, 36 h post-transfection of pCAG-JPDI-HA, HeLa cells were lysed and treated with EndoH for the indicated times, and JPDI-HA was detected by anti-HA Western blot analysis. g and d indicate glycosylated and deglycosylated forms, respectively. A nonspecific protein band is indicated by an asterisk. C, 36 h post-transfection of pCAG-JPDI-HA, HeLa cells were homogenized for preparation of microsomes. The resulting microsomes were incubated with or without proteinase K (ProK) and Triton X-100 (1% Trx-100) and subsequently subjected to anti-HA and anti-BiP Western blot analysis. A nonspecific protein band is indicated by an asterisk.

Article Snippet: After denaturing of proteins by incubation in 0.5% SDS and 1% 2-mercaptoethanol at 100 °C for 10 min, the lysates were treated with 50 mM sodium citrate (pH 5.5), endo- -N-acetylglu- at PR IN C E E D W A R D ISL A N D U N IV on N ovem ber 2, 2014 http://w w w .jbc.org/ D ow nloaded from cosaminidase H (EndoH) (a MBP-tagged version commercially called EndoHf, New England BioLabs), and 5 units/1 g of lysate protein at 37 °C.

Techniques: Transfection, Incubation, Staining, Fluorescence, Western Blot

Journal: PLoS Neglected Tropical Diseases

Article Title: Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

doi: 10.1371/journal.pntd.0004120

Figure Lengend Snippet: 100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoH f glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti- Strep -tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.

Article Snippet: Recombinant EndoH f glycosidase (EndoH f ) was from New England Biolabs, UK.

Techniques: Incubation, Staining, Western Blot, Strep-tag, Avidin-Biotin Assay, SDS Page

Journal: PLoS Neglected Tropical Diseases

Article Title: Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

doi: 10.1371/journal.pntd.0004120

Figure Lengend Snippet: Size exclusion chromatography on Superdex200 column and detection at E 280nm was used, MWs of different peaks were determined and assigned to oligomeric (1), dimeric (2) and monomeric (3) TconTS1 as described under Methods. A: Oligomerisation pattern of TconTS1 (solid line) or TconTS2 (dashed line), 300 μg protein was loaded. B: Effect of TconTS1 deglycosylation on enzyme oligomerisation. 100 μg TconTS1 were deglycosylated using 4000 units EndoH f glycosidase in phosphate buffer pH 7.4 for 4 hours and directly applied to the column (dashed line). As control 100 μg TconTS1 was treated correspondingly without the addition of enzyme was loaded (solid line).

Article Snippet: Recombinant EndoH f glycosidase (EndoH f ) was from New England Biolabs, UK.

Techniques: Size-exclusion Chromatography