Endo h

Protein samples (100 μg) were boiled in Glycoprotein Denaturing Buffer (NEB, Ipswich, MA, USA) for 10 min. For Endoglycosidase H (Endo H) treatment, samples were added to a reaction containing GlycoBuffer 3 (NEB) and Endo H (1000 units, NEB) and incubated at 37 °C for 1 h. For PNGase F treatment, samples were added to a reaction containing GlycoBuffer 2 (NEB), 1% Nonidet P-40 (NEB), and PNGase F (1000 units, NEB) and incubated at 37 °C for 1 h.

Trypsin: Cells were treated with 2 ml of 0.25% Trypsin in EDTA (Gibco, Gaithersburg, MD) at 37°C for 10 min before inactivation and protein extraction using NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris‐HCl, final pH 8.0) plus 1 μM DTT, 25 μM PMSF, and 0.1 μg/ml aprotinin and leupeptin.
Endo H: 50 micrograms of protein obtained from NP40 lysis were treated with 1,500 units of Endo H (New England Biolabs, Ipswitch, MA) at 37°C for 1 hr.

SDS-PAGE was performed in 8–10% polyacrylamide gels run under reducing or non-reducing conditions. Separated proteins were either detected by Coomassie Brilliant Blue staining or by transfer onto nitrocellulose membranes (Hybond-C, GE Healthcare) and subsequent detection with different antibodies and chemiluminescence-based detection reagents. Detection of the αC was done using a polyclonal goat anti-human alpha chain specific antibody (Sigma–Aldrich), the λC was detected using a rabbit anti-human lambda light chain antibody (Sigma–Aldrich) and the SC was detected using a rabbit anti-human SC antibody (Gentaur).
Crude protein extracts, SSL7-prufied sIgA1 or IF fractions were subjected to enzymatic deglycosylation. For endoglycosidase H (Endo H) digestion 1.5 μl of 10x Glycoprotein Denaturing Buffer (NEB, 5% SDS, 0.4 M DTT) were added to 13.5 μl of sample. This mix was incubated for 10 min at 95°C. After the sample had cooled down on ice, 2 μl G5 Buffer (NEB), 1 μl Endo H (NEB) and 2 μl ultrapure water were added and this mix was incubated for 60 min at 37°C. For the peptide: N-glycosidase F (PNGase F) digestion 1.5 μl of denaturing buffer were added to 13.5 μl of sample. This mix was incubated for 10 min at 95°C. After the sample had cooled down on ice, 2 μl G7 Buffer (NEB), 1 μl PNGase F (NEB), and 2 μl NP-40 were added and this mix was incubated for 60 min at 37°C.

Wild-type Mgst2 gene was engineered by introducing N-linked glycosylation site (Asn-Ser-Thr) followed by a linker sequence (Gly-Ser-Ala-Gly-Ser-Ala-Gly-Ser-Ala-Gly) between the residues 2Ala-3Gly. The engineered Mgst2 gene was cloned into pGEM1 vector for expression. The construct was transcribed and translated in vitro using TNT® SP6 Quick Coupled System (Promega) in the presence and absence of column-washed rough microsomes (CRM) of pancreas from dog. In all, 5 μl of reticulocyte lysate, 500 ng of plasmid DNA, 0.5 μl of [³⁵S]Met, and 0.5 μl of CRM were mixed and incubated at 37 °C for 90 min. For Endo H treatment, 9 μl of the TNT reaction was mixed with 1 μl of 10× glycoprotein denaturing buffer, 0.5 μl of Endo H (500,000 units/ml; New England BioLabs), 7.5 μl of MillQ water, and 2 μl of 10× GlycoBuffer 3. The reaction mix was incubated at 37 °C for 1 h. Translated products were analyzed by 12% SDS-PAGE after heating at 40 °C for 10 min, and the gel was visualized on Fuji FLA-3000 PhosphorImager (Fuji film) with the Image Reader 8.1J/Image Gauge software.