Fig 1: Structure and function of PStRAP(A) The structures of MFG-E8 (i), RAP (ii), and PStRAP (iii) are depicted. RAP has tetrapeptide sequence HNEL at the C terminus for retaining in the ER, and PStRAP replaces this sequence to DYKDDDDK tag. Full-length sequence of PStRAP is shown in Figure S1A. SP, signal peptide. (B) PStRAP was detected using the capillary electrophoresis immunoblotting with anti-DYKDDDDK tag antibody from the culture supernatants of murine primary hepatocytes after mRNA transfection. Gel-like image view of anti-DYKDDDDK tag antibody responses (left). Arrowhead indicates PStRAP with a predicted size of 76.4 kDa. The amounts of DYKDDDDK-tagged protein normalized to the total protein content (right). d, day; cdn opt, codon optimization. (C) Membrane lipid array was performed on the culture supernatants from the murine primary hepatocytes one day after mRNA transfection. PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol. (D) Oxaliplatin (L-OHP; 500 μM) or vehicle-pretreated MCA205 cells were stained with PStRAP and BV421-conjugated anti-DYKDDDDK tag antibody or BV421-conjugated annexin V and counter stained with 7-AAD. (E) Thymocytes pretreated with dexamethasone (Dex; 10 μM) were stained with BV421-conjugated annexin V in the presence of PStRAP (n = 3/group). Relative geometric mean fluorescent intensity (gMFI) based on the Dex untreated group is shown. The gating strategy, individual plots, and gMFI values are shown in Figure S2. Error bars show mean (SD). Significance differences were determined using one-way ANOVA followed by post hoc Tukey’s test. (F) CD91 expression level in RAW264.7 cell sublines transfected with shRNA was detected by immunoblotting (top). Pictures of the uncropped membrane are shown in Figure S3. The relative optical density was calculated using the optical density of the scramble shRNA transfectant as a reference (bottom). (G) The luciferase secreted upon NF-κB activation was measured in the culture supernatants from the RAW264.7 cell sublines 1 hour after stimulation with PStRAP or recombinant RAP protein (n = 3/group). The fold change was calculated using the luciferase level in the culture supernatants without stimulation as a reference. Error bars show mean (SD). Significance differences were determined using two-way ANOVA followed by post hoc Tukey’s test. (H) RAW264.7 was cultured with CypHer5E-labeled thymocytes that had been pretreated with Dex (10 μM) for 4 h, in conditioning medium containing PStRAP- or mock-supernatant for 2 h. For the competitive assay, either annexin V or RAP protein was added to the medium during co-culturing. The CypHer5E-positive RAW264.7 cells were detected using flow cytometer, and the phagocytosis rate was calculated (n = 3/group). The gating strategy and individual plots are shown in Figure S4. Error bars show mean (SD). Significance differences were determined using an unpaired Welch’s t test.
Fig 2: PS and CD91 recognition enable for PStRAP-induced adaptive immune responses(A, B, and E) MMC-pulsed MCA205-PStRAP, its mutant (ΔPS or ΔCD91) (A), RAP co-expressing (B), or PStRAP (Gas6) (E) transfectants (5 × 105 cells), or PBS were subcutaneously inoculated into the left flank of the mice. Viable MCA205 (5 × 105 cells) cells were subcutaneously transplanted into the right flank seven days later, and then tumor volumes were measured every other day (n = 8/group). Significance differences were determined using two-way ANOVA followed by post hoc Tukey’s test. (C) The effect of RAP on T-cell proliferation in vivo was examined. Wild-type C57BL/6 mice were adoptively transferred with CFSE-labeled OT-I cells, 1 day before immunization with MMC-pulsed MCA205-OVA with or without PStRAP and RAP expression. After three days, the draining subiliac lymph nodes were harvested and stained for TCR-Vβ5 and CD8 expression. The percentages of dividing OT-I cells are shown (n = 4/group). Error bars show mean (SD). Significance differences were determined using one-way ANOVA followed by post hoc Tukey’s test. The gating strategy and individual histograms are shown in Figure S8. (D) The structures of Gas6 (top) and PStRAP (Gas6) (bottom) are depicted. SP, signal peptide; SHBG, sex hormone-binding globulin.
Supplier Page from R&D Systems, a Bio-Techne Brand for LRPAP Protein
Available conjugates: Sizes Available: 50 ug