Disuccinimidyl suberate dss

Lipopolysaccharide (LPS) (L9641), Phorbol-12-myristate-13-acetate (PMA) (P1585), Ac-YVAD-cmk (SML0429) and Disuccinimidyl suberate (DSS) (S1885) were purchased from Sigma-Aldrich. Recombinant murine M-CSF (315–02) was purchased from PeproTech. Puromycin (abs9143) was purchased from Absin. RPMI-1640 and DMEM were obtained from Gibco. Nigericin (tlrl-nig) was obtained from InvivoGene. VX-765 (S2228) and MCC950 (S7809) was purchased from Selleck. Antibody against Flag (M185-7) (1:2000) and monoclonal mouse anti-HA (M180-7) (1:5000) were purchased from MBL. Monoclonal rabbit anti-NLRP3 (D2P5E) (1:1000), monoclonal rabbit anti-GSDMD (E9S1X) (1:1000), monoclonal rabbit anti-cleaved GSDMD (Asp276) (E3E3P) (1:1000), monoclonal rabbit anti-Caspase-1 (E2Z1C) (1:1000), monoclonal rabbit anti-Cleaved Caspase-1 (Asp296) (E2G2I) (1:1000) were purchased from Cell Signaling Technology. Monoclonal mouse anti-ASC (sc-271054) (1:1000) were purchased from Santa Cruz Biotechnology. Polyclonal rabbit anti-IL-1β (ab9722) (1:1000) were purchased from Abcam. Monoclonal anti-HRP-conjugated α-Tubulin (HRP-66031) (1:5000) was purchased from Proteintech. Human IL-1β ELISA Kit (DLB50) and Mouse IL-1β ELISA Kit (MLB00C) were purchased from R&D Systems. LDH Cytotoxicity Assay Kit (C20300) was purchased from Invitrogen.

Measurement of ferrireductase activity was performed on extracts of SH-SY5Y cells transfected to overexpress human α-syn. The stable cell line and the method to measure the activity were as previously reported [29 (link)]. Tetramers were detected by crosslinking the extracts with the crosslinker, disuccinimidyl suberate (DSS) (Sigma), according to the manufacturer’s instructions and as previously described [29 (link)]. Crosslinked species were detected with western blot, as described above, for non-crosslinked α-syn. High molecular weight aggregates of α-syn were detected, as previously described in cross linked samples of SH-SY5Y cells overexpressing α-syn [26 (link)].

BMDCs were seeded in 12-well plates (2 × 10⁶ cells/well) and treated with live H. capsulatum or LPS (100 ng/ml, 6 h) plus ATP (5 mM, 30 minutes) for 18 h. Cells were centrifuged at 4500 rpm for 5 min. Cell pellets were resuspended in cold 0.3 ml buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose) and protease inhibitor cocktail. Cell lysate was obtained after passing the suspension through 29-G syringe for 15 times. Cell lysates were spun at 4500 rpm for 8 min to remove contaminating nuclei, unlysed cells and H. capsulatum. Supernatants were diluted with 1 volume of CHAPS buffer (20 mM HEPES-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA and 0.1% CHAPS) to lyse residual organelles before centrifugation at 8000 rpm for 8 min. After centrifugation, supernatants were discarded and the pellets were treated with disuccinimidyl suberate (DSS, 2 mM, Sigma-Aldrich) for 30 min at room temperature. The cross-linked pellets were re-suspended in 40 μl SDS sample buffer and proteins were separated on 12.5% SDS polyacrylamide gel followed by immunoblotting with anti-ASC antibody (Adipogen) as previously described [45 ]

To determine the relationship between ZmGa1P and ZmPME10-1, fraction 8 of pollen secretory proteins of SDGa25 and J66 pollen was desalted with a PD MiniTrap G-10 column (GE) in 50 mM hydroxyethyl piperazineethanesulfonic acid buffer (pH 8.0) and concentrated in a filtrate column (Amicon, Millipore). The conditioned samples were cross-linked by adding disuccinimidyl suberate (DSS, Sigma) at a 75-fold molar excess. After incubation for 1 h at room temperature, the reaction was quenched by adding 1 M Tris (pH 8.0) to a final concentration of 50 mM. The samples were separated on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and subjected to western blotting using the antibody against ZmGa1P at 1:500 dilution. For mass spectrometry analyses, the ~100-kD bands separated by SDS-PAGE were stained with CBB and excised for LC-MS/MS analyses as described above. This experiment was also performed using the total proteins extracted from unpollinated and pollinated SDGa25 silks. Three biological replicates were tested. Western blots for crosslinking proteins were shown in Supplementary Figure 21.

For immunoblotting, the equal amounts of total proteins were subjected to SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies (Table S2) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Vector Laboratories, Burlingame CA, USA) (Table S2). Immunoreactive protein detection was performed with an enhanced chemiluminescence detection system (GE Healthcare, Pittsburgh, PA, USA).
For ASC oligomerization assay, resultant pellets from cells were washed with cold 1X PBS, crosslinked with 4 mM disuccinimidyl suberate (DSS, Sigma-Aldrich, St. Louis, MO, USA) for 30 min, and pelleted by centrifugation. The crosslinked pellets were resuspended by the sample buffer for immunoblotting analysis.