Alexa fluor 568 conjugated goat anti mouse igg antibodies

Cells were transfected with either WT or variant METTL23-FLAG vectors using ViaFect transfection reagent (Promega) on 24-well coverslips (MS-92132, Sumitomo Bakelite) in 24-well plates. After fixation with 4% (PFA) and permeabilization with 0.3% Triton X-100 in PBS for 10 minutes, the transfected cells were blocked with protein block serum-free (Dako) for 1 hour and incubated with the primary antibody, anti-METTL23 antibody (1:500; Thermo Fisher Scientific; PA5-71814), and anti-FLAG antibody (1:500; CST; 9A3) overnight at 4°C. Then, Alexa Fluor 568–conjugated goat anti–mouse IgG antibodies (1:500; Invitrogen, Thermo Fisher Scientific), Alexa Fluor 488–conjugated goat anti–rabbit IgG antibodies (1:500; Invitrogen, Thermo Fisher Scientific), and DAPI (1:500; Dojindo) were used to detect FLAG, METTL23, and DNA signals, respectively. The cells were mounted with Ultramount Aqueous Permanent Mounting Medium (DakoCytomation) and visualized under a confocal fluorescence microscope (LSM700; Zeiss).

Cos-7 cells (4 × 10⁴) were plated on glass coverslips on 24-well plates. Cells were transfected with 0.5 µg of empty vector or expression vectors for wild-type MFSD2a or N-glycosylation mutants using PEI (1 µg DNA: 7 µL PEI ratio). Forty-eight hours after transfection, cells were fixed with 2% PFA in PBS for 15 min at RT and then washed three times in PBS. Cells were permeabilized for 15 min in PBS 0.01%/Triton X-100 at RT and washed three times in PBS. Blocking solution (5% FBS and 5% BSA in PBS) was added for 45 min at RT, and, after addition of mouse monoclonal anti-V5 antibodies (1/500, R960-25, Thermo Fisher Scientific), cells were incubated overnight at 4 °C with shaking. Following three washes in PBS, cells were incubated with Alexa Fluor 568-conjugated goat anti-mouse IgG antibodies (1/1000, A11004, Invitrogen, Thermo Fisher Scientific) for 1½ h at RT. Cells were then washed three times in PBS, treated with DAPI NucBlue Cell Stain ReadyProbes reagent (Thermo Fisher Scientific) for 45 min at RT, and finally mounted on a drop of ProLong Gold antifade reagent (Invitrogen, Thermo Fisher Scientific) after three washes in PBS. Signals were analyzed by confocal microscopy using the Eclipse Ti microscope coupled with an A1R confocal unit and the CFI Plan Apochromat λ 60×/1.4 objective (Nikon Canada, Mississauga, ON, Canada).

Transfected cells were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.1% Triton X-100 for 5 min. Samples were washed three times with 1% PBST, followed by blocking with protein block serum-free (Dako) for 1 h and incubation with ANTI-FLAG M2 (1:500; F1804, SIGMA)/anti-firefly luciferase antibody (1:200; ab21176, Abcam) overnight at 4 °C. Following three washes with PBST, the samples were incubated with Alexa Fluor 568-conjugated goat anti-mouse IgG antibodies (1:500; Invitrogen)/Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies (1:500; Invitrogen) and DAPI (1:500; Dojindo) for 1 hour at RT to visualize the antigens and nuclei. These were mounted with Ultramount aqueous permanent mounting medium (DakoCytomation) and visualized under a confocal fluorescent microscope (LSM700, Zeiss).