Fig 2: The analysis of the detailed interactions between the JL2 Fab and SCARB2. (A) Five polypeptide elements, three from the heavy chain (purple) and two from the light chain (marine), surround the domain III of SCARB2. Ten residues, S28, T30, G31, Y32, Y54, Y55, R100, S102, Y105, and Y107, in the heavy chain and four residues, D31, Y32, Y50, and I55, in the light chain interact with SCARB2 (with distance less than 4.5 Å). The residues in JL2 Fab involved in the interactions with SCARB2 are shown as sticks. (B) Similar to (A), a semi-transparent surface rendering of SCARB2 presents the detailed interactions. All residues involved in the interactions are shown as sticks

Fig 3: EGF co-localizes with LAMP1 at 30 mai. (A) Co-localization of EGF with M6PR, LAMP1 or SCARB2 is shown by immunofluorescence of EGF and M6PR, LAMP1 and SCARB2. RD-hSCARB2 cells were infected with EV71 and then fixed 30 min after the addition of EGF followed by immunofluorescence. DAPI is in blue, EGF is in green, whereas organelle markers are in red. The panels on the right are enlarged 3D cross-section views of the dashed rectangles in the merged panels. Arrowheads indicate co-localization of EGF with markers. Representative images are shown. Scale bar: 10 µm. (B) Quantification of co-localization of EGF with specific markers. Number of z stack sets was two except SCARB2 was three. ns, not significant, **P<0.01. Statistical significance was determined by a two-tailed, unpaired t-test. Error bars represent s.d.

Fig 4: The monoclonal antibody JL2 binds human SCARB2. (A) Cells overexpressing human SCARB2 and SCARB2 knockout cells were lysed and analyzed by Western blot using the anti-human SCARB2 polyclonal antibody (pAb) (top panel) and JL2 (middle panel). The lowest panel: β-actin was used as a loading control. (B) Surface staining of 293-hSCARB2 cells. Open area represents staining with JL2 and shaded area represents isotype control staining. (C) The binding of 293-hSCARB2 cells with different concentrations of JL2 (square) and the IgG2a isotype control (circle)

Fig 5: The complex structure of JL2 Fab bound to SCARB2 (at pH 7.0). (A) The overall complex structure. The JL2 Fab binds to the head of SCARB2 (Domain III) at a perpendicular angle with helices α2, α5, and α14 involved in the interaction. Domain I, II, and III of SCARB2 are colored in orange, violet and yellow, respectively. Glycans are shown as sticks. (B and C) Cartoon representation of the interacting residues in SCARB2 (B) and Fab (C). The residues in SCARB2 involved in the interactions with JL2 Fab are shown as sticks (B). The residues in JL2 Fab involved in the interactions with SCARB2 are highlighted in bright colors and labeled on the surface (C)