Fig 2: The identified HULC interacting proteins.a The volcano plot of the identified proteins. The proteins significantly enriched in the tobramycin purified samples are shown as red dots. Log2 fold change was plotted on the x-axis and −log10 P value was plotted on the y-axis. b The protein interactome network of HULC. PPI information was obtained through a database search using String 9.0 and imported into Cytoscape 3.1.1 for network construction. Proteins and their interactions are shown as nodes and edges. Node size reflects the interaction degree. Proteins are grouped based on their known biological functions. c Validation of selected identified proteins by RIP. Immunoblots of TGM2, DDX58, LGALS3BP, H2A and PDIA3 in the cell lysates and immunoprecipitates are shown in the upper panel. The agarose gel electrophoresis images of HULC amplified by qRT-PCR are shown in the lower panel. LincRNA-p21 was used as an irrelevant lncRNA control. See also Supplementary Figs. 2 and 3. Source data are provided as a Source Data file.

Fig 3: TGM2 promotes BV2 activation independent of its transamidase-activity. (a) Western blotting analysis of the overexpression efficiency of TGM2-WT, TGM2-C277S, and TGM2-R580K in BV2 cells. (b) The effects of TGM2-WT, TGM2-C277S, and TGM2-R580K on the mRNA expression of inflammatory genes (Il1b, IL6, Tnf, Ptgs2, and Nos2) in BV2 cells were analyzed by real-time qPCR analysis (n = 3). (c) The effects of TGM2-WT, TGM2-C277S, and TGM2-R580K on the level of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in conditioned medium from BV2 cells were analyzed by ELISA (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig 4: Propofol attenuates NF-κB signaling to inhibit microglial activation. (a) Western blotting analysis of p-NF-κB and NF-κB levels in LPS-primed BV2 cells upon treatment with propofol and/or overexpression of TGM2-WT and TGM2-R580K; β-actin was loaded as a control. (b) The effects of propofol and/or TGM2-WT/TGM2-R580K on NF-κB activity in LPS-primed BV2 cells were analyzed by luciferase reporter assay (n = 3). (c) The effects of propofol and/or TGM2-WT/TGM2-R580K on the mRNA expression of inflammatory genes (Il1b, IL6, Tnf, Ptgs2, and Nos2) in LPS-primed BV2 cells were analyzed by real-time qPCR analysis (n = 3). (d) The effects of propofol and/or TGM2-WT/TGM2-R580K on the level of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in conditioned medium from LPS-primed BV2 cells were analyzed by ELISA (n = 3). (e) Western blotting analysis of the effect of constitutively active IKKβ (CA-IKKβ) on the p-NF-κB and NF-κB levels in LPS-primed BV2 cells with propofol treatment. (f) The effects of CA-IKKβ on the NF-κB activity in LPS-primed BV2 cells with propofol treatment were analyzed by luciferase reporter assay (n = 3). (g) The effects of CA-IKKβ on the mRNA expression of inflammatory genes (Il1b, IL6, Tnf, Ptgs2, and Nos2) in LPS-primed BV2 cells with propofol treatment were determined by real-time qPCR analysis (n = 3). (h) The effects of CA-IKKβ on the level of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in conditioned medium from LPS-primed BV2 cells with propofol treatment were analyzed by ELISA (n = 3). p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig 5: TGM2 activates NF-?B signaling to promote microglial activation. (a) The effects of TGM2 knockdown on the activation of NF-?B signaling in LPS-primed BV2 cells were analyzed by Western blotting. (b) The effects of TGM2 inhibition with GK921 on the activation of NF-?B signaling in LPS-primed BV2 cells were analyzed by Western blotting. (c) The effects of TGM2 knockdown on the NF-?B activity in LPS-primed BV2 cells were analyzed by luciferase reporter assay. (d) The effects of TGM2 inhibition with GK921 on the NF-?B activity in LPS-primed BV2 cells were analyzed by luciferase reporter assay. (e) The effects of Bay11-7082 (10 µM) on the mRNA expression of inflammatory genes (Il1b, IL6, Tnf, Ptgs2, and Nos2) in LPS-primed BV2 cells were analyzed by real-time qPCR analysis (n = 3). (f) The effects of Bay11-7082 (10 µM) on the level of inflammatory cytokines (IL-1ß, IL-6, and TNF-a) in conditioned medium from LPS-primed BV2 cells were analyzed by ELISA (n = 3). *p < 0.05 and ***p < 0.001.