Mouse anti ha 11 antibody

The following materials were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 NL4.3 infectious molecular clone (pNL4.3), from Dr. Malcolm Martin [61 (link)]; and TZM-bl cells, from Dr. John C. Kappes and Dr. Xiaoyun Wu [62 (link)–66 (link)]. Rabbit polyclonal anti-Nef antiserum was obtained from the NIBSC Center for AIDS Reagents program (cat. # ARP444, from Dr M Harris). Mouse anti-HA.11 antibody was purchased from Biolegend (clone 16B12) and rabbit polyclonal anti-SERINC3 antiserum was purchased from Abcam (cat # ab65218). pX330-U6-Chimeric_BB-CBh-hSpCas9, used for gene editing of SERINC3, was obtained from Addgene (plasmid #42230, gift from Dr. Feng Zhang) [67 (link), 68 (link)].

To determine whether the anti-hACE2 mAbs induced hACE2 internalization, live A549 cells expressing hACE2 receptor with an HA-epitope tag appended to its intracellular C-terminus were incubated with anti-hACE2 mAbs (1 μg ml⁻¹) for 1 h at 37 °C. Then, cells were fixed with 4% paraformaldehyde/PBS, treated with 10 mM glycine and permeabilized with 0.1% Triton X-100. Total hACE2-HA was then detected with mouse anti-HA.11 antibody (BioLegend, cat 901503, clone 16B12, 1 μg ml⁻¹). The internalization of the hACE2-HA protein and anti-hACE2 mAbs was then evaluated by staining with goat anti-mouse Alexa Fluor 594 (to detect the HA-tagged hACE2) (1/500 dilution) and/or goat anti-human Alexa Fluor 488 antibodies (to detect the anti-hACE2 mAbs) (1/500 dilution) (Thermo Fisher Scientific). As a control, anti-CD44 antibody conjugated with FITC (clone F10-44-2 from abcam, cat. no. ab30405, 1/20 dilution) was used. Wheat germ agglutinin conjugated with Alexa Fluor 594 (Thermo Fisher Scientific, cat. no. W11262, 5 μg ml⁻¹) were used to visualize the cell surface. Images were captured using a DeltaVision OMX SR imaging system (GE Healthcare).