Endoh

Twenty-four hours in advance, HeLa cells were transfected with a mix of polyethylenimine (pEI) and WT or mutant DNA in a ratio of 2.5 to 1, after which the cells were subjected to pulse-chase analysis as described before [23] (link). In short, cells were starved in medium lacking cysteine and methionine for 15–30 min and pulse labelled for 10 min with 55 µCi/35 mm dish of Express ³⁵S protein labelling mix (PerkinElmer, Boston, MA). The pulse was stopped and the chase started by the first of 2 washes with chase medium containing an excess of cold cysteine and methionine. At the end of each chase time, cells were cooled on ice and further disulfide bond formation and isomerization was blocked with 20 mM iodoacetamide (IAM).
Cells were lysed and detergent lysates were subjected to immunoprecipitation with polyclonal antibody 40336 against gp160 or with CD4-IgG2 against the CD4-binding site. Next, samples were deglycosylated using EndoH (Roche) as described above, and were subjected to non-reducing and reducing (25 mM DTT) 7.5% SDS-PAGE [23] (link), [24] (link). Gels were dried and exposed to Kodak MR films for autoradiography.

U373-NKG2DL cells were lysed in PBS 1%NP-40 and HALT protease inhibitor (Thermo Fisher) followed by SDS-PAGE protein separation and transfer to PVDF. Immunoblotted proteins were detected with ECL2 (Thermo Scientific). For Pulse-chase experiments, U373-NKG2DL cells were starved for 1 h in DMEM minus cysteine (cys) and methionine (met) supplemented with 10% FBS (pulse media). After starve period, EXPRE³⁵S³⁵S Protein Labeling Mix (PerkinElmer) was added to pulse media at 150 μCi/10⁶ cells for 30 min. Cells were chased for the indicated times then lysed, digested, and immunoprecipitates were separated by SDS-PAGE and detected by autoradiography. Digestions using EndoH (Roche) or PNGaseF (NEB) were performed according to manufacturer’s instructions.

CHO cells were tranduced with either Ad-tTA (25 MOI) or Ad-197 (20 MOI) and Ad-tTA (5 MOI). At 24 h post transduction (p.t.), the cells were washed with PBS, overlaid with DMEM (Cys⁻/Met⁻), and incubated for 1.5 h. The cells were pulsed with 300 µCi/10⁶ cells for 45 min and the label was chased for the indicated time intervals. CHO cells were washed with ice-cold PBS and lysed with ice-cold PBS-1% NP-40 buffer. Cell lysates were pre-cleared with agarose beads and immunoprecipitated with αFLAG Ab conjugated to agarose beads (Sigma-Aldrich, St. Louis, MO). The samples were eluted from the beads with 50 mM NaOAc-0.15% SDS buffer (10 min, 98°C) and treated with EndoH (Roche Diagnostics, Indianapolis, IN) or PNGase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocols. The samples were separated on a 6% polyacrylamide gel.

Purified rCLCA1 was treated for 3 h at 37 °C with PNGaseF (Roche Applied Science) and a combination of sialidases (SialEXO cleaves α2–3, α2–6, and α2–8; Genovis, Lund, Sweden) and O-glycosidase (New England Biolabs) after denaturation in 0.1% SDS at 95 °C for 5 min and subsequent addition of Triton X-100 to 1% final concentration.
For EndoH analysis, lysates from transfected cells were reduced in sample buffer containing DTT (100 mm) at 95 °C for 5 min. The pH was adjusted to 5.5 by adding sodium citrate. After addition of EndoH (Roche) or PBS as control, the samples were incubated at 37 °C overnight before analysis by SDS-PAGE and immunoblotting.

Membrane fractions prepared from transiently transfected HEK293 cells were solubilised in 1% Triton X-100 and 0.1% SDS in phosphate buffered saline (PBS; 150 mM NaCl, 2.6 mM KCl, 10 mM Na₂PO₄, 1.9 mM KH₂PO_4, pH 7.4). Samples were centrifuged (21,255×g, 15 min, 4 °C) to remove debris. Endoglycosidase H (EndoH, 5 mU/100 µl of sample) or endoglycasidase F (EndoF; 1 U/100 µl of sample; Roche Diagnostics GMBH, Mannheim, Germany) were added to equal aliquots of solubilised membrane samples and incubated overnight at 37 °C [40 (link)] before immunoblot analysis.

To investigate glycosylation status of heterologously expressed human tetraspanins in S. cerevisiae, a sample of crude isolated yeast membranes containing tetraspanin-GFP fusion proteins were treated with endoglycosidase H (EndoH). Yeast membranes were mixed with EndoH (Roche) and 9x Buffer (0.25 M sodium citrate) and thereafter incubated for 2 h at 37°C. Samples were then loaded on SDS-PAGE, as described above, and investigated using in-gel fluorescence.