Endoglycosidase h

Leaf protein (5 µg) samples containing H1-HFBI were diluted in 50 mM sodium citrate pH 5.5 (HCl), 0.5 mM PMSF. Then, 0.2 U/ml Endoglycosidase H (Roche) was added. After incubation at 37°C for 0, 15, or 60 min, the reaction was stopped by the addition of SDS loading buffer.

Bloodstream form trypanosomes were cultured in the presence of tunicamycin
(Sigma) added to complete media at 1 µg/ml, which prevents further cell
proliferation 52 (link). For treatment with
PNGase F, 1 x 10⁸ cells were washed in PBS and incubated with lysis
buffer (1% NP-40, 100 mM NaPO₄, pH 7.5) plus protease inhibitor
cocktail (Roche) and then heated to 95˚C for 15 minutes. Samples were treated
with 10 mU PNGase F (NEB) overnight at 37˚C. A second aliquot of PNGase F was
added and the reaction continued for 2 hours prior to analysis. For treatment
with Endoglycosidase H, 1 x 10⁸ cells were washed and lysed by
incubation with 1 ml of 5 mM EDTA and protease inhibitor cocktail (Roche),
followed by two cycles of freeze-thawing. Samples were centrifuged to separate
crude membranes and the pellet dissolved in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA,
1% SDS. 5 mU/ml of Endoglycosidase H (Calbiochem) was added to each sample, and
then 5 mU/ml again 12 hours prior to analysis.

Cell expressions of different C-terminally tagged Atg15 constructs were grown to stationary phase, approx. 60 OD₆₀₀ units, were harvested (3000 rpm, RT, 5 min), washed twice with cold TBS (20 mM Tris/HCl pH 7.6, 200 mM NaCl) and resuspended in 400 µL lysis buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% sodium desoxycholate, 2% Triton X-100, 0.1% [w/v] SDS, 1 mM PMSF, Complete proteinase inhibitor Mix [Roche]) and vortexed with 400 µL glass beads at 4 °C for 30 min. Cell debris was removed by centrifugation (1× at 500× g, 4 °C, 5 min; 2× at 13,200 rpm, 4 °C, 5 min). The supernatant was first incubated with 3 µL anti-HA antibody (0.2 mg/mL) for 3 h at 4 °C on a rotating wheel and then 1 h with 6 mg protein A-sepharose. The samples were centrifuged, washed twice with lysis buffer and once with washing buffer (50 mM KH₂PO₄ pH 5.5, 0.02% [w/v] SDS) and resuspended in washing buffer supplemented with 0.1 M β-mercaptoethanol. One half of each sample was incubated with 15 mU endoglycosidase H (Roche) for 1 h at 37 °C, then 6 µL of 6× SDS sample buffer was added, and the samples were analyzed using SDS-PAGE and Western blotting.

Endoglycosidase H (Roche, Basel, SUI) treatments were performed on ML-DmBG3-c2 cells using NEB (Ipswitch, MA, USA) protocols and buffers. Pelleted cells were resuspended in lysis buffer (Guy, 2000 (link)) [10 mM Hepes pH 7.6,1.5 mM MgCl2, 10 mM KCl, 250 mM sucrose, 5 mM EGTA, 5 mM EDTA, Protease Inhibitor (Calbiochem, San Diego, CA, USA)], flash frozen, thawed on ice, homogenized, and spun down (1000× g, 5 min). Supernatant was transferred to new tubes, mixed with 10×Glycoprotein denaturing buffer, and put at 100°C for 10 min. This denatured solution was transferred to new tubes and 10×G5 was added to make a 1× and 1% NP40 final concentration. Endoglycosidase H was then added and tubes put at 37°C for 4 h, after which reactions were quenched with 6× sample buffer (βME+Protease Inhibitor) and prepared for western blots.

VHHs fused to the natural llama heavy-chain antibody long hinge region, and a hexahistidine (his6) tag was produced in baker’s yeast using vector pRL188 and purified from culture supernatant using immobilized-metal affinity chromatography as described earlier [23 (link)]. VHHs produced in this manner were indicated by the suffix “F.” About 10% of the VHH amount produced in this manner was dimerized through the single cysteine present at the C-terminus of the VHH, immediately preceding the his6 tag. Both monomeric and dimeric VHHs are useful for functional immobilization of VHHs to polystyrene surfaces by passive adsorption [9 (link),24 (link)]. Yeast-produced VHHs were biotinylated at a weight ratio of protein to biotin of 5 using amine-reactive sulfo-N-hydroxysuccinimide-LC-biotin (ThermoFisher Scientific).
VHHs were subjected to reducing SDS-PAGE using precast gels (Novex, San Diego, CA, USA), and stained using GelCode Blue reagent (ThermoFisher Scientific). VHHs were digested with endoglycosidase H (Roche Applied Science) according to the manufacturer’s instructions.

All enzymes, as well as plasmid pGEM1, TNT T7 Quick Coupled System and rabbit reticulocyte lysate were from Promega (Madison, WI, USA). ER rough microsomes from dog pancreas were from tRNA Probes (College Station, TX, USA). EasyTag™ EXPRESS³⁵S Protein Labeling Mix, [³⁵S]-L-methionine and ³⁵S-L-cysteine, for in vitro labeling was purchased from Perkin Elmer (Waltham, MA, USA). Restriction enzymes and Endoglycosidase H were from Roche Molecular Biochemicals (Basel, Switerland). Proteinase K was from Sigma-Aldrich (St Louis, MO). The DNA plasmid, RNA clean-up and PCR purification kits were from Thermo Fisher Scientific (Ulm, Germany). All oligonucleotides were purchased from Macrogen (Seoul, South Korea).