Mab3126

Transfected cell lysate supernatants were incubated with mouse monoclonal anti-hFcRL3/FcRH3 (MAB3126, R&D Systems), rat monoclonal anti-hFOLR4 (JUNO) (MAB6328, R&D Systems), or rabbit polyclonal anti-hIZUMO1 ABIN6059408 (antibodies online) primary antibodies overnight at 4°C on a rocking platform. Isotype controls used appropriate antibodies of mouse, rat, or rabbit IgGs (Sigma-Aldrich). With added agarose, protein A/G beads (Thermo Fisher Scientific) were incubated at RT for 2 hours. A negative control of protein A/G agarose beads alone was also prepared. Coimmunoprecipitates (Co-IPs) were eluted from protein A/G agarose beads by incubation in the reducing sample buffer for 5 min at 95°C. After electrophoretic separation in 10% polyacrylamide gel and transfer onto a PVDF membrane, the FcRL3 Co-IP was incubated with rat monoclonal anti-hFOLR4 (JUNO) antibody (MAB6328, R&D Systems); IZUMO1 Co-IP was incubated with mouse antibody against FcRL3/FcRH3 (MAB3126, R&D Systems) diluted 1:500 in 5% milk overnight at 4°C. After washing and incubation with secondary anti-rat or anti-mouse antibody conjugated with HRP (Bio-Rad) diluted 1:3000 in 5% milk, the antibody reaction was visualized using the SuperSignal Chemiluminescence Substrate (Thermo Fisher Scientific).

Nonreducing lysis buffer (2× SDS) was used for whole-cell lysates. Using the Mem-PER Plus kit, membrane and cytosol protein fraction were isolated. Protein was quantified using a NanoDrop 3000 spectrophotometer (Thermo Fisher Scientific). Molecular masses were estimated with prestained Precision Plus Protein Dual Color Standards (Bio-Rad). Tris-glycine buffer (pH 9.6) with 20% methanol was used for transfer of proteins onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Darmstadt, Germany) for electroblotting carried out for 1 hour at 500 mA. PVDF membranes blocked with 5% dry milk (Bio-Rad) in PBS-Tween were washed and incubated with primary antibody mouse monoclonal anti-hFcRL3/FcRH3 (MAB3126, R&D Systems) or rat monoclonal anti-hFOLR4 (JUNO) (MAB6328, R&D Systems), both diluted 1:500 in 5% dry milk (Bio-Rad) in PBS-Tween, overnight at 4°C. A mouse monoclonal anti–α-tubulin (TU02) (sc8035, Santa Cruz Biotechnology) diluted 1:5000 in 5% dry milk (Bio-Rad) in PBS-Tween was used as a loading control. Incubation with secondary antibody anti-mouse or anti-rat IgG conjugated to horseradish peroxidase (HRP; Bio-Rad), both diluted 1:3000 in PBS-Tween, was performed for 1 hour at RT. After washing, membranes were developed using the SuperSignal Chemiluminescence Substrate (Thermo Fisher Scientific).