endoglycosidase treatment  (New England Biolabs)

Journal: eLife

Article Title: Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody

doi: 10.7554/eLife.36217

Figure Lengend Snippet: Expression and functionality of the P2X7-EGFP constructs in HEK cells and expression of the transgene in mice. ( A ) The EGFP sequence was fused via a Strep-tag-His-tag linker to the very C-terminus of the mouse P2X7 sequence. (B/C) Protein extracts from transiently transfected (Lipofectamine 2000, Invitrogen) HEK cells (DSMZ (ACC 305), regularly tested for mycoplasma contamination) were separated by SDS-PAGE with endoglycosidase treatment as indicated. Gels were analyzed by western blotting with a P2X7-specific antibody (Alamone, extracellular) ( B ) or by direct EGFP-fluorescence scanning ( C ). ( D ) Normalized dose–response curves for ATP-induced ethidium uptake. HEK293 cells were cultured and transfected (2 μg DNA/well of a six-well-plate, Lipofectamin, Thermo Fisher Scientific). After 27 hr, cells were seeded in 96-well plates (5 × 10 4 cells/well) and incubated in the presence of 20 μM ethidium bromide in PBS for 15 min. Dye influx was evaluated with a fluorescence plate reader (Fluostar Galaxy, BMG) upon addition of the indicated ATP concentrations, as described ( Bruzzone et al., 2010 ). Lines represent nonlinear curve fits of the Hill equation to the data and were normalized to the calculated maximal responses. EC 50 values are 582 (CI 498–681), 840 (CI 644–1098) and 582 (CI 457–740) for wt (L variant) and EGFP-tagged L and P variants, respectively. Error bars represent SEM (n = 4–7). CI = 95% confidence interval. ( E ) Patch-clamp recordings from HEK cells transfected as above with wt and EGFP-tagged P2X7 (L variants). Recordings were performed as described ( Nicke et al., 2009 ) in normal or low divalent cation (DIC) containing extracellular solution to account for possible unspecific effects ( Nörenberg et al., 2016 ). Representative current traces from n > 3 cells are shown. Due to problems with the perfusion, one trace is incomplete (1 mM ATP for P2X7-EGFP in low DIC). ( F ) Southern blot controls for correct modification and integrity of the P2X7 BAC clone during subsequent homologous recombination steps. ( G ) Reproducibility and stability of endogenous and transgenic P2X7 protein expression. Protein extracts from four line 17 mice were separated by SDS-PAGE and quantified by western blotting and infrared imaging using antibodies against P2X7 (Synaptic Systems) and vinculin and fluorescent secondary antibodies. Data are presented as mean ±SD from four individual mice. No significant difference of endogenous P2X7 expression was found in two independent experiments.

Article Snippet: 10ml of extract were separated by SDS-PAGE with with or without endoglycosidase treatment (30 min at 37 °C in the presence of reducing loading buffer (1x) and 5 IUB miliunits EndoH or 10 IUB miliunits PNGaseF (New England Biolabs)).

Techniques: Expressing, Construct, Mouse Assay, Sequencing, Strep-tag, Transfection, SDS Page, Western Blot, Fluorescence, Cell Culture, Incubation, Variant Assay, Patch Clamp, Southern Blot, Modification, BAC Assay, Homologous Recombination, Transgenic Assay, Imaging

Journal: PLoS ONE

Article Title: Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)

doi: 10.1371/journal.pone.0063230

Figure Lengend Snippet: GlyT2 expression after a pulse-chased in culture cells. COS7 cells transfected with GlyT2 cDNA in pCDNA3 were pulse-labeled for 15 min with [ 35 S]methionine/cysteine and chased for the times indicated in the conditions given in Material and Methods. The cells were then surface biotinylated, lysed and the protein lysate was either immunoprecipitated with GlyT2 antibody (total transporter, A) or bound to streptavidin-agarose and sequentially immunoprecipitated with GlyT2 antibody (biotinylated fraction, B). Proteins extracted from the beads were resolved in SDS-PAGE. (A) Kinetics of GlyT2 expression. (B) Kinetics of GlyT2 plasma membrane expression in the same cells as in A. Lower panels: (A) densitometry of the 100 kDa and 75 kDa bands in the fluorograms. (B) Biotinylated bands are represented as a percentage of each of the lanes labeled in A. Bars represent the S.E.M. (n = 3). (C) Cells were treated overnight with the vehicle alone (DMSO) or with 10 µg/ml tunicamycin, and then pulse-chased as described above. (D, E) Immunoprecipitates were treated overnight with the vehicle alone (endoglycosidase buffer, -) or with the indicated endoglycosidase (+) in denaturing conditions, and then resolved by SDS-PAGE as described in the Materials and Methods. The transporter proteins (100 kDa, 75 kDa and 60 kDa) are indicated with arrowheads.

Article Snippet: Carbohydrate Modification Pulse-chased GlyT2 immunoprecipitates were digested with the desired endoglycosidase (PNGase F, New England Biolabs; or Endoglycosidase H or D, Roche) in a small volume of the appropriate buffer, following the manufacturer’s instructions.

Techniques: Expressing, Transfection, Labeling, Immunoprecipitation, SDS Page