Rabbit anti eea1 antibody

Cells were seeded into 48 well plates with a density of 3.0 × 10⁴ cells/well. After incubation overnight and medium change, FAM-labelled or dual-labelled nucleosomes were added to the medium (final 20 nM) and incubation was continued for 12 h. After washing with PBS twice, cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized for 10 min in 0.3% (v/v) Triton X-100 and blocked with 5% BSA for 30 min at room temperature. Cells were incubated with rabbit anti-EEA1 antibody (Abcam, Cat #109110, diluted 1:500) or anti-MB21D1(cGAS) rabbit anti-mouse antibody (ABclonal, Cat #8335, diluted 1:100) overnight at 4 °C. After washing with PBS twice, cells were incubated with the secondary antibody, Cy3 goat anti-rabbit IgG (Abclonal, Cat #AS007, diluted 1:100) for 1 h, and then, nuclei were stained with DAPI for 7 min. Image capture was performed using an OLYMPUS FV1000S-IX81 laser scanning confocal microscope.

Cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes and treated for 10 minutes with PBS containing Triton X-100 (0.25%). The samples were then incubated with mouse IgG–blocking reagent (BioLegend), followed by the addition of goat anti-NCF1 antibody (1:50, Abcam) and rabbit anti-EEA1 antibody (1: 100, Abcam) for 1 hour and then washing. AF488 donkey anti–goat IgG antibodies (1:100) were then stained for 30 minutes and washed. Finally, AF647 goat anti–rabbit IgG antibodies (1:100) were incubated for 30 minutes at room temperature. To detect the colocalization of NCF1 and LAMP1, AF647 rat-mouse antibody (1:100, BioLegend) and a goat anti-NCF1 antibody (1:50, Abcam) were incubated for 1 hour and washed. Then, AF488 donkey anti–goat IgG antibody (1:100) was stained for 30 minutes at room temperature. After that, cells were incubated with DAPI (1:5000, BD Biosciences) for 5 minutes. Cells were then detached onto glass dishes and viewed and photographed on a confocal fluorescence microscope (THUNDER Imager Tissue and Leica TCS SP8, Leica Microsystems). All immunofluorescence was assessed in a blinded manner by 2 observers throughout the analysis process. The colocalization of NCF1 and EEA1 and of NCF1 and LMAP1 was calculated with ImageJ software (NIH).

Sections of the paraffin-embedded cell pellets and the breast cancer tissues were prepared and processed for antigen retrieval as above. They were processed for IHF as described previously [13 (link)]. After being blocked with 10% goat serum for 20 min at room temperature, they were incubated with anti-γ1-adaptin antibody (BD Bioscience; 1:1000) alone for cell pellets, or with a mixture of anti-γ1-adaptin antibody and either rabbit anti-EEA1 antibody (Abcam, Cambridge, UK; 1:200 dilution) or sheep anti-human TGN46 (Bio-Rad Laboratories, Inc., CA, USA, 1:400 dilution) for patient specimens, for 1 day at 4 °C. They were then incubated with Alexa 594-conjugated donkey anti-mouse IgG (1:800 dilution), or a mixture of Alexa 594-conjugated donkey anti-mouse IgG and Alexa 488-conjugated donkey anti-rabbit IgG for 60 min at room temperature. They were observed with a confocal laser microscope (FLUOVIEW FV1000, OLYMPUS, Japan).