Western lightning chemiluminesce nce plus

Purified IgA1 was isolated by 10% SDS-PAGE under reducing or nonreducing conditions and then transferred to a PVDF membrane (GE Healthcare). The membrane was blocked with 3% BSA in PBS containing 0.1% Tween 20 (PBS-T), followed by incubation with the anti-IgA1 mAb (1∶1000) and a peroxidase-conjugated anti-mouse IgG (1∶5000, GE Healthcare). For lectin blot analysis, HRP-conjugated WFA (1∶1000) or jack bean lectin concanavalin A (ConA, 1∶4000) were used. Cross-reacting bands were detected using Konica immunostaining kit (Konica, Tokyo, Japan) for anti-IgA1 mAb and ConA and Western Lightning Chemiluminesce nce Plus (Perkin-Elmer, Boston, MA) for WFA.

Sodium orthovanadate (Na₃VO₄), trypsin inhibitor, PMSF, Nonidet P-40, Triton X-100, formalin 10%, aprotinin and leupeptin were obtained from Sigma-Aldrich Canada (Oakville, ON, Canada) and the Western Lightning Chemiluminescence Plus from PerkinElmer (Guelph, ON, Canada). Fetal bovine serum (FBS) and Dulbeco’s modified Eagle’s medium (DMEM) were purchased from Wisent Bioproducts (St-Bruno, QC, Canada). And Tween 20 as well as hydrogen peroxide (30%) from Fischer Scientific (Ottawa, ON, Canada). Polyethylenimine (PEI) was obtained from VWR (Mississauga, ON, Canada) and slowFade^TM Gold antifade reagent from Thermo Fisher Scientific (Waltham, MA, USA).