Fig 1: The activation of MAPK signaling pathway was essential for FMDV replication. (A and B) PK-15 cells were mock infected or infected with FMDV at an MOI of 0.5 for 0, 2, 4, 8, 12, or 16 h. The cells were then lysed and subjected to Western blotting. (A) The expression of RPSA and VP1 proteins was detected by using anti-RPSA and anti-VP1 antibodies, respectively. (B) The expression levels of MAPK pathway related proteins, including JNK1/2, ERK1/2, and p38 kinases and phosphorylated JNK1/2, ERK1/2, and p38 kinases (p-JNK1/2, p-ERK1/2, and p-p38), were detected by Western blotting. (C) PK-15 cells were pretreated with DMSO (solvent control) or 20 µM U0126 for 1 h and then infected with FMDV for 8 h. The expression levels of p-ERK1/2 and FMDV VP1 proteins were detected by Western blotting. (D and E) PK-15 cells were pretreated with DMSO or U0126 for 1 h and then infected with FMDV for 0, 4, or 8 h. (D) The expression levels of p-ERK1/2 and FMDV VP1 proteins were detected by Western blotting. (E) The mRNA expression levels of CCL2, CCL5, IL-6, TNF-a, and FMDV were measured by qPCR.
Fig 2: Expression of ribosomal protein SA (RPSA) in polymorphonuclear neutrophil (PMN) stimulated by SS2. (A) RPSA expression on bone marrow-derived neutrophils induced by SS2 was detected by flow cytometry. APC indicates Ly-6G and FITC indicates RPSA. (B) RPSA expression on bone marrow-derived neutrophils induced by SS2 was observed by laser confocal microscopy. (C) RPSA expression on bone marrow-derived neutrophils was quantified by qPCR. *p < 0.05, **p < 0.01, compare with control group, ns, no significant difference.
Fig 3: Detection of the polymorphonuclear neutrophil (PMN) and bacterial load in the brain after infected with SS2. (A) Pathological changes in brain tissue were observed by HE staining. The scale bar represents 100 µm (top), 400 µm (bottom). (B, C) Immunohistochemistry was used to observe the pathological changes in the brain tissues of infected mice. The scale bar represents 100 µm (top), 400 µm (bottom). (D) The infiltration of neutrophils in the brain homogenates of mice (n=5) was detected by flow cytometry. (E) The expression of ribosomal protein SA (RPSA) on the surface of neutrophils in brain homogenates was detected by flow cytometry. (F) Brain tissue bacterial load. Five mice were used in each group for each experiment. *p < 0.05, **p < 0.01, ***p < 0.001 compare with control group, ns, no significant difference.
Fig 4: LAMR1 recruits EIF3S5 to deubiquitinate ZIKV E protein. (a) HEK293T cells were co-transfected with pHA-LAMR1 and pFlag-USP13, pFlag-USP15, pFlag-USP26, pFlag-USP30, pFlag-USP38, pFlag-USP49, pFlag-OTUB1, pFlag-EIF3S5, or pFlag-BRCC3. Lysates were prepared and used for IP with an anti-Flag antibody and analyzed by SDS-PAGE. (b) HEK293T cells were co-transfected with pHA-E, pMyc-UB, pFlag-USP13, and pFlag-EIF3S5. Lysates were prepared and used for IP with an anti-HA antibody and then analyzed by SDS-PAGE. (c) EIF3S5-knockdown HEK293T cells and control cells were co-transfected with pFlag-E, pMyc-UB, and pHA-LAMR1. Lysates were prepared and used for IP with an anti-Flag antibody and then analyzed by SDS-PAGE. (d) LAMR1-knockdown HeLa cells and control cells were co-transfected with pFlag-E, pMyc-UB, and pHA-EIF3S5. Lysates were prepared and used for IP with an anti-Flag antibody and then analyzed by SDS-PAGE. (e, f) HEK293T cells were transfected with plasmids encoding Flag-EIF3S5 and HA-LAMR1. Cell lysates were prepared with lysis buffer and then analyzed by IP with the indicated antibodies. (g, h) HEK293T cells were transfected with plasmids encoding Flag-EIF3S5 and HA-E. Cell lysates were prepared with lysis buffer and then analyzed by IP with indicated antibodies and immunoblotting as described above. (i) HEK293T cells were transfected with plasmids encoding Flag-EIF3S5 and GFP-LAMR1, GFP-LAMR1 (1–85aa), and GFP-LAMR1 (86–259aa). Cell lysates were prepared with lysis buffer and then analyzed by IP with the indicated antibodies. (j, k) HeLa cells stably expressing sh-EIF3S5 or control sh-RNA were generated and analyzed. Cells were transfected with pHA-LAMR1 or empty vector for 16 h, and then infected with ZIKV (MOI = 1) for 48 h. The levels of viral protein and RNA were detected by immunoblotting and qPCR, respectively
Fig 5: Flow cytometry detection of SS2 phagocytosis by RPSA+ PMN and RPSA- PMN. (A, B) Phagocytosis of fluorescent microspheres by RPSA+ PMN and RPSA- PMN. X axis represents microspheres and Y axis represents RPSA for bottom panel. (C, D) Phagocytosis of fluorescent microspheres by RPSA+ PMN and RPSA- PMN after blocking ribosomal protein SA (RPSA). *p < 0.05, ***p < 0.001 compare with control group, ns, no significant difference.
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