Fig 1: The interactions among CXCL12, KRT19, and TGM2. (A) Biotinylated human CXCL8 or CXCL12 was immobilized on SA beads, which were incubated with KRT19. The bead-bound proteins were eluted with SDS and subjected to SDS-PAGE and immunoblotting with SA-HRP or anti-KRT19 antibody. (B) SA beads bearing CXCL12–biotin were incubated with increasing concentrations of KRT19. Bound KRT19 was detected by SDS-PAGE and immunoblotting with anti-KRT19 antibody, and intensities of the KRT19 bands were measured. (C) CXCL12–biotin that was immobilized on SA beads was incubated at 4 °C with TGM2 in the absence or presence of KRT19 and in the absence or presence of 10 mM CaCl2. The bound proteins were eluted and subjected to SDS-PAGE and immunoblotting with SA-HRP and antibodies to KRT19 and TGM2. (D) SA beads bearing preformed complexes of CXCL12–biotin and KRT19 were incubated at 20 °C for 15 min with TGM2 in the absence or presence of the TGM2 inhibitor ERW1041E. The proteins were eluted from the beads and detected by SDS-PAGE and immunoblotting with antibodies to CXCL12 and KRT19.
Fig 2: Nepenthes fluid proteases degrade crude gliadin and reduce antigenic load.(a) Digestion profile of α-gliadin peptide 33mer at 20:1 (substrate:enzyme), using fluid proteases under dilute conditions. Normalized relative abundance measured from LC-MS ion chromatograms, using caffeine as internal standard. (b–d) SDS-PAGE of 10 mg/ml gliadin digested with the indicated concentration of (b) pepsin, (c) fluid proteases or (d) pepsin in combination with fluid proteases. Digestions at pH 2.5 and 37 °C, for 90 min. All concentrations in μM. M: Molecular weight markers, and gliadin: total crude gliadin in the absence of protease. (e) Weighted average peptide length as a function of fluid protease concentration, with or without pepsin, using ion chromatogram intensities for weighting. Peptide data from LC-MS/MS runs, with intensities obtained using Protein Deconvolution 1.0. (f) Barcode representation of deconvoluted spectra from (e), at the noted enzyme concentrations, showing molecular weight distribution with simple binary model of intensities, (white: <0.2%, black: >0.2%). (g) TG2-induced conversion of gliadin digestion products to deamidated counterparts. Fractional deamidation quantified for all peptides from ion chromatogram intensities; data presented as a ratio, where the antigenic peptide deamidation is normalized to the non-antigenic peptide deamidation at the indicated protease concentration. Antigenic regions defined using the DQ2 criteria1139, inset table.
Fig 3: Responses of hepatic metastases formed with control, Krt19-edited, or Tgm2-edited PDA cells to treatment with anti–PD-1 antibody. (A and B) Sections of hepatic metastases formed with luciferase-expressing sgScramble control PDA cells, sgKRT19-edited (A), or sgTGM2-edited (B) PDA cells were stained with fluorochrome-conjugated antibodies to KRT20 or KRT19 to reveal cancer cells and with CD3 to reveal T cells. The distance of each cancer cell from the nearest T cell was calculated for entire cross-sections, and the distance distribution of the sgScramble control tumors and their matching sgKRT19 or sgTGM2 tumors were plotted. (Scale bars, 100 µm.) More than 4,000 cancer cells from two to seven tumor sections of each group were analyzed; ****P < 0.0001, Kolmogorov–Smirnov test. (C) Mice bearing hepatic metastases formed with these luciferase-expressing PDA cells were treated with nonimmune IgG or anti–PD-1 IgG (arrows), and growth of the metastases was measured by bioluminescent imaging.
Fig 4: The immunological responses of tumors formed with Tgm2-edited mouse PDA cells. sgScramble control or sgTGM2-edited PDA cells were inoculated s/c in wild-type hosts or TGM2-deficient hosts. (A) Sections of each of the four tumor types were stained with fluorochrome-conjugated antibodies to KRT19, CXCL12, and CD3. Representative images are shown. (Scale bars, 100 µm.) (B) The growth curves of each of the four tumor types are shown; n = 5. (C) Growth curves are shown of tumors formed with sgScramble control PDA cells and sgTGM2-edited PDA cells, respectively, in wild-type hosts that were untreated or treated with nonimmune IgG or T cell–depleting antibodies to CD4 and CD8; n = 5. (D) The mRNA levels of immune genes in tumors formed with sgTGM2-edited PDA cells in wild-type hosts were compared to those of tumors formed with sgScramble control PDA cells; n = 6; mean ± SEM; ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test.
Supplier Page from R&D Systems, a Bio-Techne Brand for Transglutaminase 2/TGM2 Protein
Available conjugates: Sizes Available: 50 ug