Goat anti mouse igg secondary antibody

Cell and tissue proteins were extracted as previously described (19 (link)), and bicinchoninic acid method was used to determine the protein concentration. Nitrocellulose membranes were incubated with primary antibodies (anti-NLRP3, anti-IL-18, GAPDH, Proteintech Group, Inc., Chicago, IL,USA; anti-GSDMD, Abbexa Ltd, Cambridge, United Kingdom; anti-caspase-1, anti-IL-1β, anti-cleaved caspase-1, anti-cleaved IL-1β, Affinity Biosciences, Cincinnati, OH, USA; anti-caspase-11 p20, Santa Cruz Biotechnology, Inc.Dallas, Texas, USA) at 4 °C overnight. The membranes were washed with 1% TBST before and after incubation with goat anti-rabbit IgG secondary antibody (LI-COR Biotechnology, Lincoln, NE, USA) or goat anti-mouse IgG secondary antibody (LI-COR Biotechnology, Lincoln, NE, USA) for 1 h at room temperature. Odyssey CLx imaging system (LI-COR Biosciences, Lincoln, NE, USA) was used to analysis protein expression as previously described (19 (link)).

The following antibodies were used in this study: anti-centromere antibody (ACA; or CREST-ImmunoVision; HCT-0100), anti-Myc (Roche; 11667203001), anti-Smc1 (Bethyl; A300-055A), anti-Rpb1 (Abcam; ab5408), anti-H3K4me2 (EMD Millipore; 07–030), anti-H3K9me3 (Abcam; ab8898), anti-Actin (Invitrogen; MA5-11869), anti-CENP-B (Abcam; ab25734), anti–CENP-A (EMD Millipore; 07–574), and anti–Rpb2-pSer2 (BioLegend; H5). Anti-Sgo1, anti-Bub1, and anti-Wapl were made in-house as described previously (Liu et al., 2013a (link); Qu et al., 2019 (link)). Anti-Sororin antibodies described previously were a gift from Dr. Susannah Rankin at Oklahoma Medical Research Foundation, Oklahoma City, OK (Liu et al., 2013b (link)).
The secondary antibodies were purchased from LI-COR: Goat anti-Mouse IgG Secondary Antibody (926–68070) and Goat anti-Rabbit IgG Secondary Antibody (926–32211).
For immunoblotting, primary and secondary antibodies were used at 1-µg ml⁻¹ concentration.

Protein degradation was assessed as previously described [31 (link)], with minor differences. Briefly, strains were grown in liquid YPAD overnight at 30 °C with shaking, then diluted to a starting OD₅₉₅ of 0.75 and grown for 1hr prior to adding CHX (50 µg/mL final concentration; VWR International, LLC., Radnor, PA, USA, product #97064-724). After the treatment period, optical density was used to approximate cell concentrations and normalize the total amount of cells harvested within each strain to the least-dense culture. Cells were harvested and lysed as described in [8 (link),31 (link)]. 17 µL of lysate were loaded onto a polyacrylamide gel, transferred to a PVDF membrane, and blotted with the appropriate primary antibody. LCD-Sup35 fusions were probed with an anti-Sup35C monoclonal antibody (E4 [68 (link)], a gift from Susan Liebman), LCD-GFP fusions were probed with an anti-GFP monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallax, TX, USA, product #sc-9996), and native G-rich and Q/N-rich proteins possessing a 2×HA tag were probed with an anti-HA monoclonal antibody (BioLegend, San Diego, CA, USA, product #901502). For all blots, a fluorophore-conjugated, polyclonal goat anti-mouse IgG secondary antibody was used (LI-COR, Lincoln, NE, USA, product #926-32210).