Journal: Communications Biology

Article Title: Receptor for advanced glycation end-products (RAGE) mediates phagocytosis in nonprofessional phagocytes

doi: 10.1038/s42003-022-03791-1

Figure Lengend Snippet: a Left panel: Internalization of spores in HEK293 cells expressing the indicated RFP fusions or RFP alone. Right panel: Western blotting analysis using an anti-RFP antibody to verify the expression of the RFP fusions. Actin is used as a loading control. b Internalization of spores in HEK293T (wt) or HEK293T RAGE KO cells expressing the indicated RFP fusions. c Internalization of latex beads in wild-type HEK293T or HEK293T RAGE KO cells. The indicated polynucleotide-bound latex beads were incubated with wild-type HEK293T or HEK293T RAGE KO cells. d Top panel: Purified RAGE 1–341 -His-FLAG was subjected to SDS-PAGE analysis and stained with Coomassie brilliant blue. Bottom panel: Quantification of fluorescence intensities of Cy3-RNA precipitated with RAGE 1–341 -His-FLAG attached to agarose beads or bare agarose beads (control). e Left panel: Binding of RAGE 1–341 -His-FLAG to tRNA-bound latex beads. RAGE 1–341 -His-FLAG was incubated with tRNA-bound beads or bare beads (bead), and the protein precipitated with the beads were detected with western blotting analysis using an anti-His tag antibody. Right panel: Quantification of RAGE 1–341 -His-FLAG precipitated with tRNA-bound beads or bare beads (bead). Relative intensities of RAGE 1–341 -His-FLAG precipitated with the beads were shown. The intensity of RAGE 1–341 -His-FLAG precipitated with bare beads was defined as 1. f The internalization process of spores in HEK293T cells followed by time-lapse microscopy. HEK293T cells transiently expressing RAGE-GFP were preincubated with LysoTracker Red (lysosome) and were then incubated with spores. Representative images of a spore being internalized (arrow) are shown. Scale bar, 10 µm. Data were presented as the mean ± SEM ( a – e ). Statistical significance was determined by two-tailed unpaired Student’s t -tests. n = 3. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns not significant ( P ≥ 0.05).

Article Snippet: The following primary antibodies were used: rabbit anti-RAGE (Abcam, 1: 2000); rabbit anti-SYK (Abcam, 1: 3000); rabbit anti-RFP (InvivoGen, 1:3000); mouse anti-GFP (TransGen Biotech, 1:3000); rabbit anti-GLUT1 (Abcam, 1:4000); mouse anti-actin (TransGen Biotech, 1:3000); mouse anti-His (TransGen Biotech, 1:5000).

Techniques: Expressing, Western Blot, Incubation, Purification, SDS Page, Staining, Fluorescence, Binding Assay, Time-lapse Microscopy, Two Tailed Test