Supersignal chemiluminescence substrate

Cells were lysed to obtain whole-cell lysates as described previously.19 (link) Cell lysates were electrophoretically separated using 7.5% or 10% gels. Proteins were transferred to nitrocellulose membranes (Protran; Schleicher and Schuell BioScience Inc, Keene, NH, USA). COX-2 and iNOS proteins in cell lysates and tissue homogenates were detected using a monoclonal anti-COX-2 antibody (Cell Signaling Technology Inc, Danvers, MA, USA) and anti-iNOS (NOS-2) antibody (Santa Cruz Biotechnology Inc, Dallas, TX, USA). Signals were visualized using the SuperSignal chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA). Experiments were performed in duplicate, and equivalent loading was confirmed by probing the blots anti-α-tubulin antibody (Santa Cruz Biotechnology).

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).

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).

Proteins from HUVECs were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% PMSF (Beyotime, Shanghai, China) and quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-μm PVDF membranes (Millipore, Billerica, MA, USA), and incubated overnight at 4 °C with specific antibodies: anti-p16, anti-p21, anti-FIS1, anti-SAHH (Abcam, Cambridge, UK), anti-p53, anti-Drp1, anti-OPA1, anti-MFN1, anti-MFN2, anti-DNMT1, anti-DNMT3A, anti-DNMT3B, and anti-GAPDH (Cell Signaling Technology, Danvers, MA) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA). The dilution ratio was 1:1000 for the primary antibodies and 1:10,000 for the secondary antibodies. Protein bands were visualized with Super Signal Chemiluminescence Substrate (Thermo Scientific, Waltham, MA, USA) and GAPDH was used as a control. Images were captured using the FluorChem E system (Protein Simple, Minneapolis, MN, USA) and band densities were quantified using image J software (NIH, Bethesda, MD, USA). The densities of the bands were normalized to that of GAPDH.