RAB27A Antibody - #DF6702

Fig. 4 CD45 overexpressing tumor cells were resistant to T cell killing. a T cell killing effect was tested in DLD1 cells that had been indirectly pre-cocultured with Jurkat cells. For the evaluation of T cell killing, PBMCs (effector; E) were incubated with CD45− or CD45+ DLD1 cells (tumor cell; T) at a E:T ratio of 2:1. b T cell cytotoxicity assay of DLD1 cells (preincubated with PBS or Jurkat cell-derived EVs) in co-culture with PBMCs after the addition of OKT3 (1 μg/mL) at various E:T ratios. The images on the left are representative images of survived DLD1 cells stained with crystal violet, and the graph on the right is a line graph showing the relative intensity of survived DLD1 cells. c, d Immunoblot analysis showing CD45 expression after CRISPR Cas9 knockout of PTPRC or RAB27A knockdown in Jurkat cells. e Bar graph showing EVs-derived CD45 levels from supernatant of Jurkat cells by ELISA assay. f T cell cytotoxicity assay of DLD1 cells (preincubated with PBS or Jurkat cell-derived EVs (EVs from Jurkat-Ctrl, Jurkat-sgCD45 or Jurkat-shRAB27A cells) in co-culture with PBMCs after the addition of OKT3 (1 μg/mL) at different E:T ratios. g Schematic diagram showing the different CD45 isoforms tested in the study. h Immunoblot analysis of DLD1 cells overexpressing different CD45 isoforms. i Immunofluorescence imaging of membrane CD45-GFP on DLD1 cells. Scale bar = 5 μm. j Comparison of CD45 expression on DLD1 cells and Jurkat cells. k Proliferation assay of DLD1 cells overexpressing different isoforms of CD45. l, m T cell cytotoxicity assay of DLD1 cells with or without CD45 overexpression co-cultured with PBMCs in the presence of pre-coated OKT3 (1 μg/mL) at different E:T ratios. n–p Quantitative analysis of IFN-γ, TNF-α, and IL-2 secretion in the co-culture system of T cell cytotoxicity assay with DLD1 cells. q Proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled CD8+ T cells in the co-culture system of T cell cytotoxicity assay with DLD1 cells. r Immunoblot analysis of Caco2 cells overexpressing different CD45 isoforms. s, t T cell cytotoxicity assay of Caco2 cells with or without CD45 overexpression co-cultured with PBMCs in the presence of pre-coated OKT3 (1 μg/mL) at different ratios. u–w Quantitative analysis of IFN-γ, TNF-α, and IL-2 secretion in the co-culture system of T cell cytotoxicity assay with Caco2 cells. x Proliferation of CFSE-labeled CD8+ T cells in the co-culture system of T cell cytotoxicity assay with Caco2 cells. y NK cell sorting strategy from WBCs. z Lysis percentage of NK cell killing Caco2 cells with density diluted NK cells at NK: Tumor ratios of 80: 8 to 5: 8. All bar graph data are presented as means ± SEM. *P 

FIGURE 6 Intra-articular injection of FLS-targeting AAV for delivering Rab27a-shRNA to specifically reduce EVP secretion. (A) Rab27a expression levels in FLSs after transfection with control shRNA and Rab27a knockdown shRNA. (B, C) EVP size distribution and quantification after transfection with control shRNA and Rab27a knockdown shRNA. ** Indicates p < 0.01, versus the indicated groups, student’s t-test. (D) Representative Western blot images for positive and negative surface markers of EVPs at equal concentration after shRNA transfection. (E) Schematic diagram of constructing a virus targeting FLS to inhibit Rab27a for intra-articular injection. The synovium-affinity peptide HAP-1 is fused with the AAV9 viral capsid viral protein 2 (VP2), and the AAV9 vector is designed to simultaneously carry the gene encoding the mScarlet fluorescent protein and an expression cassette for shRNA. (F) Representative images of mScarlet fluorescence in the synovium and cartilage regions of mouse knee joints. Scale bar = 200 µm and 100 µm separately. (G) Representative images of mScarlet fluorescence in sections of multiple mouse organs. Scale bar = 200 µm. (H) Representative images and quantitative results of RAB27A expression in the synovial region after intra-articular AAV injection. ** Indicates p < 0.01, versus the indicated groups, two-way ANOVA.

Figure 2 Icariin regulated MITF/Rab27A in osteoclasts. (A) Molecular docking between icariin and MITF. In the 3D plot, the blue solid line represents the hydrogen bond, the gray dotted line represents the hydrophobic force, in the 2D plot, the pink solid line represents the hydrophobic force, and the green and light green dotted lines both represent the hydrogen bond. (B) Representative images of immunofluorescence staining of MITF (green). The nucleus was stained with DAPI, and cytoskeleton were stained with phalloidin. (C) The histogram represents the relative positive expression of MITF in each group. **P < 0.01 compared with the control groups. (D) Representative images of immunofluorescence staining of Rab27A (green). The nucleus was stained with DAPI, and cytoskeleton were stained with phalloidin. (E) The colocalization Pearson correlation coefficient (PCC) of Rab27A and cytoskeleton. Each dot represents the PCC per view under the microscope, and mean is indicated in black dotted lines. *P < 0.05, **P < 0.01 compared with the control groups. (F and G) Western Blot was used to test the protein expressions of MITF and Rab27A in osteoclasts treating with icariin, *P < 0.05, compared with the control groups.

Figure 2 Icariin regulated MITF/Rab27A in osteoclasts. (A) Molecular docking between icariin and MITF. In the 3D plot, the blue solid line represents the hydrogen bond, the gray dotted line represents the hydrophobic force, in the 2D plot, the pink solid line represents the hydrophobic force, and the green and light green dotted lines both represent the hydrogen bond. (B) Representative images of immunofluorescence staining of MITF (green). The nucleus was stained with DAPI, and cytoskeleton were stained with phalloidin. (C) The histogram represents the relative positive expression of MITF in each group. **P < 0.01 compared with the control groups. (D) Representative images of immunofluorescence staining of Rab27A (green). The nucleus was stained with DAPI, and cytoskeleton were stained with phalloidin. (E) The colocalization Pearson correlation coefficient (PCC) of Rab27A and cytoskeleton. Each dot represents the PCC per view under the microscope, and mean is indicated in black dotted lines. *P < 0.05, **P < 0.01 compared with the control groups. (F and G) Western Blot was used to test the protein expressions of MITF and Rab27A in osteoclasts treating with icariin, *P < 0.05, compared with the control groups.