Ab69639

Mouse Trim59, Trim27, WASH, and control siRNAs were purchased from Ribo, Shenzheng, China. Anti- Trim59 (ab69639, Abcam), anti-Trim27 (A6405, Abclonal), anti-WASH (SAB-4200552, Sigma), anti-Oct4 (ab18976, Abcam), anti-FLAG (M20008, Abmart), anti-HA (#T501-1, Signalway Antibody), anti-Ub (YT4793, Immunoway), anti-K48 (#4289, Cell Signaling Technology), anti-K63 (BML-PW0600, Enzo), anti-ACTR2 (ab134082, Abcam), anti-MYH9 (ab138498, Abcam), anti-CAPZA1 (ab166892, Abcam), anti-CAPZB (ab175212, Abcam), anti-β-actin (SC-81178, Santa), Phalloidin-iFluor 488 (ab176753, Abcam), DAPI (#4083, Cell Signaling Technology), Alexa Fluor 488 Conjugate (#4416, #4408, Cell Signaling Technology), and Alexa Fluor 594 Conjugate (#8890, #8889, Cell Signaling Technology) antibodies were purchased.
Murine FL Trim59 clone was obtained from the ATCC. Trim59 mutants were constructed by performing PCR with four primers according to a previous method^{33 (link)}. All primers used in this study are listed in supplementary Table S2b. YFP–WASH and YFP-WASH K220R mutant were from Patrick Ryan Potts, UT Southwestern Dallas, TX 75390, USA; plasmids encoding hemagglutinin (HA)-tagged ubiquitin (HA-Ub), HA-K48-Ub, and HA-K63-Ub were obtained from Y. Xiong (University of North Carolina, Chapel Hill).

HMC3 cells were lysed in RIPA lysis buffer (Beyotime). A 1/10 volume of supernatant was collected as the input, and half of the remaining supernatant was incubated with 20 µL/mL protein A/G Sepharose beads (Beyotime) at 4 °C for 1 h to remove nonspecific hybrid proteins. Afterwards, the lysates were incubated with 2 µg anti-NLRP3 antibodies (ab263899, Abcam)/anti-TRIM59 antibodies (ab69639, Abcam) or negative control IgG (Beyotime) at 4 °C overnight and then rotated at 4 °C with a mixture of protein A/G Sepharose beads (20 µL/mL) for 4 h. The bound proteins were analysed by western blotting.

To determine the expression of galectin‐9 in HaCaT and cervical cell lines, we performed an immunocytochemistry (ICC) and immunofluorescence assay (IF). A protocol suggested by Abcam for ICC and IF was followed. HaCaT, SiHa and HeLa cell lines were cultured in an eight‐well Chamber Slide™ system (C7182; Merck) and fixed with 100% methanol for 5 min at room temperature. Nonspecific antibody binding was blocked with 1% BSA in PBS for 1 h. Then cells were incubated overnight at 4 °C with an anti‐galectin‐9 antibody (ab69639; Abcam) diluted 1 : 400 with 1% BSA in PBS. Next, cells were incubated for 1 h at room temperature with anti‐rabbit IgG (Alexa Fluor® 488) (ab150077; Abcam) diluted 1 : 1000 in 1% BSA in PBS. Nuclei staining was performed with 0.1 µg·mL⁻¹ DAPI Stain (4083; Cell Signaling Technology, Danvers, MA, USA). An isotype IgG control was applied as a negative control test. Fluorescence intensity was determined using the program zen 2.6 Lite from Zeiss (Oberkochen, Germany). The ratios of cytoplasmic and nuclear level fluorescence were also determined for each cell line.