Wild-type (WT) and Rnf128-deficient mice on a C57BL/6 background were generously provided by Dr. Chengjiang Gao (Shandong University, Jinan, China) [13]. Genotyping of WT and Rnf128−/− mice was performed with primers: forward primer, 5′-GCTGAAGTTAGTACTGCATG-3′ and reverse primer 5′-GTCAACAGGTGGCAGATACC-3′. The mice were housed in specific pathogen-free (SPF) conditions at the Experimental Animal Center of Gannan Medical University, with access to sterile water and food. Mice aged over 8 weeks, matched for age and sex, were utilized for all experiments. The Institutional Research Ethics Committee of Gannan Medical University approved this study.

HEK293 and HeLa cells were obtained from the American Type Culture Collection and cultured in DMEM (Biological Industries) supplemented with 10% FBS (Biological Industries). Human TF-1 cells, generously provided by Procell Life Science and Technology, were cultured in RPMI-1640 (Corning) medium supplemented with 2 ng/mL GM-CSF and 10% FBS. To obtain and culture bone marrow-derived macrophages (BMDMs), mice were sacrificed, and femurs and tibia were aseptically removed. Bone marrow cells were then flushed from the femur and tibia and cultured in 150-mm dishes with 15 mL of IMDM medium supplemented with 10% FBS and 20 ng/mL M-CSF for a duration of 6 days. All cells were incubated in a cell culture chamber at 37 °C with 5% CO2. Transient transfection for HEK293 and HeLa cells were performed using polyethylenimine (PEI; Polysciences, Warrington, PA, USA) as previously described [17].

Plasmids expressing Flag- or HA-tagged RNF128 for mammalian expression were generated using standard molecular cloning methods. The pcDNA3.1-IL-3Rα-Flag and pcDNA3.1-IL-3Rβ-Flag were obtained from Changsha You Bao Biotechnology Co. Ltd. (Changsha, China). Ubiquitin and its mutant ubiquitin plasmids (K6, K11, K27, K29, K33, K48 and K63) were a gift from Dr. Liang-Guo Xu (Jiangxi Normal University, Nanchang) [18, 19]. The mutant ubiquitin plasmids (K6R, K11R, K27R, K29R, K33R, K48R and K63R) were purchased from MiaoLingPlasmid (Wuhan, China). Human RNF128-shRNA constructs were generated by cloning the following double-stranded oligonucleotides into the pLKO.1 vector. The designed shRNA sequences targeting RNF128 included: scramble shRNA, 5′-AATGCACGCTCAGCACAAGC-3′; RNF128 shRNA, 5′-AGAGACTGCTGTTCGAGAAAT-3′.

Primary antibodies used in this study included Anti-IL-3Rα antibody (ABclonal, A3926), anti-STAT5 antibody (Cell Signaling Technology, 94205), anti-phospho-STAT5 antibody (Cell Signaling Technology, 4322), anti-β-actin antibody (Proteintech Group, 66009-1-Ig), anti-RNF128 antibody (Cell Signaling Technology, 71,590), anti-LC3B antibody (Cell Signaling Technology, 3868), anti-Flag-tag antibody (Sigma-Aldrich, F1804), anti-HA-tag antibody (Invitrogen, 26,183), and anti-Myc-tag antibody (Proteintech Group, 16286-1-AP) were employed. Secondary antibodies included HRP conjugated goat anti-rabbit IgG (Boster Bio, BA1054), HRP conjugated goat anti-mouse IgG (Boster Bio, BA1050), TRITC conjugated goat anti-mouse IgG (Boster Bio, BA1089), DyLight®488 conjugated goat anti-mouse IgG (Boster Bio, BA1126), TRITC conjugated goat anti-rabbit IgG (Boster Bio, BA1090), and DyLight®488 conjugated goat anti-rabbit IgG–488 (Boster Bio, BA1127). All antibodies were purchased from the respective suppliers as indicated. Recombinant human IL-3 (Pepro Tech, 200-03), mouse Il-3 (MCE, HY-P7062), mouse GM-CSF (Pepro Tech, 213–13) and LPS (Sigma-Aldrich, L2880) were purchased from the respective suppliers. MG132 (HY-13259), Chloroquine (HY-17589 A), and Cycloheximide (HY-12320) were obtained from MCE (MedChemExpress).

Total RNA extraction was carried out using TransZol-Up (TransGen Biotech, ET111-v2) and resuspended in RNase-free water following the manufacturer’s protocols. For cDNA synthesis, 1 µg of total RNA was reverse transcribed into complementary DNA using PrimeScript™ RT Master Mix (RR036A, Takara). Gene expression levels were analyzed by real-time qPCR using a SybrGreen I-based kit (TransGen Biotech, AQ601-02-V2) with gene-specific primers. The housekeeping gene GAPDH served as the internal reference to normalize for variations in RNA input and cDNA synthesis efficiency. The threshold cycle (Ct) values were determined for each sample and the ΔΔCt method was utilized for the quantitative analysis of relative gene expression. Primer sequences used in mice are as follows: Pim1-F: 5’-TGTCTCTTCAGAGTGTCAGC-3’, Pim1-R:5’-CGGATTTCTTCAAAGGAGGG-3’; Cd69-R: 5’-GTAGCAACATGGTGGTCAG-3’; Id1-F: 5’-AACTCGGAGTCTGAAGTCG-3’, Id1-R: 5’-GACACAAGATGCGATCGTC-3’; Il6-F: 5’-TAGTCCTTCCTACCCCAATTTCC-3’, Tnfα-F: 5’-CCCTCACACTCAGATCATCTTCT-3’, Tnfα-R: 5’-GCTACGACGTGGGCTACAG-3’; Cd69-F: 5’-TCTCATTGCCTTAAATGTGGG-3’, Il6-R: 5’-TTGGTCCTTAGCCACTCCTTC-3’; Kc-F: 5’-CTGGGATTCACCTCAAGAACATC-3’, Kc-R: 5’-CAGGGTCAAGGCAAGCCTC-3’; Ccl2-F: 5’-TTAAAAACCTGGATCGGAACCAA-3’, Ccl2-R: 5’-GCATTAGCTTCAGATTTACGGGT-3’; Ccl5-F: 5’-GCTGCTTTGCCTACCTCTCC-3’, Ccl5-R: 5’-TCGAGTGACAAACACGACTGC-3’; Cxcl10-F: 5’-CCAAGTGCTGCCGTCATTTTC-3’, Cxcl10-R: 5’-GGCTCGCAGGGATGATTTCAA-3’; Gapdh-F: 5’-AGGTCGGTGTGAACGGATTTG-3’, Gapdh-R: 5’-TGTAGACCATGTAGTTGAGGTCA-3’. Primer sequences used in human cells are as follows: CD69-F: 5’-GCTGGACTTCAGCCCAAAATGC-3’, CD69-R: 5’-AGTCCAACCCAGTGTTCCTCTC-3’; PIM1-F: 5’-TCTACTCAGGCATCCGCGTCTC-3’, PIM1-R: 5’-CTTCAGCAGGACCACTTCCATG-3’; ID1-F: 5’-GTTGGAGCTGAACTCGGAATCC-3’, ID1-R: 5’-ACACAAGATGCGATCGTCCGCA-3’; GAPDH-F: 5’-AGCCTCAAGATCATCAGCAATG-3’, GAPDH-R: 5’-ATGGACTGTGGTCATGAGTCCTT-3’.

Immunofluorescence staining was performed as previously described [17]. Briefly, HeLa cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and subsequently blocked by 3% BSA for 2 h to prevent nonspecific binding. Incubation with primary antibodies against the target proteins was carried out overnight at 4 °C, followed by appropriate fluorophore-conjugated secondary antibodies before fluorescence microscopy. Nuclei were visualized by counterstaining with DAPI, and imaging was conducted using a laser scanning confocal fluorescence microscope (ZeissLSM880), equipped with filters suitable for the respective fluorophores.

Co-IP and Immunoblotting experiments were performed as previously described [18, 20]. Briefly, 60 µL of protein A agarose resin (Yeasen Biotechnology) was incubated for 12 h with 0.4 µg of anti-Flag antibody for each immunoprecipitation and cell lysate by RIPA at 4 °C. After incubation, the agarose resin was subjected to wash with ice-cold lysis buffer. The immunoprecipitated samples were then analyzed using immunoblotting with the indicated antibodies. For immunoblotting, cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (PMSF, cocktail). Subsequently, protein samples were separated via 12% SDS-PAGE and electrophoretic transferred onto PVDF membranes (Millipore). These membranes were then blocked with 5% nonfat milk for 1 h, followed by an overnight incubation with primary antibodies at 4 °C. After thorough washing, the membranes underwent a 1-hour incubation with secondary antibodies. Protein visualization was achieved using an ECL chemical fluorescence chromogenic solution from Thermo Scientific, and the results were analyzed using ImageJ software.

Transwell chambers (Corning) were used for Transwell migration assay to measure the migratory response of BMDMs. Single-cell suspension of BMDMs were seeded in the upper chamber of transwell (pore size: 8 μm) at a density of 1 × 105 cells in 200 µL culture medium. The lower chamber contained culture medium with or without the addition of LPS or LPS/Il-3. After 36 h of incubation, cells were fixed in 4% paraformaldehyde, stained with 0.2% crystal violet. Non-migratory cells on the upper chamber were gently removed with a cotton swab. Transwell inserts were observed and photographed under a microscope, and the results were quantified using Image J software.

Human TF-1 cells (~ 4 × 106) were infected with either pCDH-HA-RNF128 or pCDH lentivirus by gently centrifugation at 800 rpm for 90 min. The culture media was replaced with fresh growth medium 24 h after infection and the cells were harvested at 48 h post-infection. Cell lysis was achieved by RIPA buffer for 30 min on ice and centrifuged at 12,000 × g for 8 min at 4 °C. The supernatant was incubated overnight with anti-HA agarose beads at 4 °C. The next day, beads were washed by cold 1 M NaCl RIPA buffer for 10 min and three times with PBS to remove nonspecific proteins. Proteins were solubilized by resuspending in SDS loading buffer and boiled for denaturation. Subsequently, the eluted proteins in the supernatant were analyzed by LC-MS/MS.

Statistical analysis was performed using GraphPad prism 9.0 (GraphPad Prism, San Diego, CA, USA), and the presented data are expressed as the mean ± standard error of the mean (SEM). To assess the statistical significance of variances between two groups, an unpaired Student’s t-test was employed, while differences among multiple groups were evaluated through ANOVA analysis. A significance level of *P < 0.05 was employed to denote statistical significance, indicating a noteworthy difference in the observed results.