Fig 1: CAPS affects the phosphorylation of PLK1 by interacting with MYPT1. (A) Venn diagram showing 27 genes found to interact with CAPS, among which two genes were involved in the G2/M phase transition. (B,C) Co‐IP assay showing the interaction of CAPS with MYPT1, but not with RPS27A. (D) Phosphorylation at different sites of MYPT1 in LN229 and GBM1 cells expressing control shRNA, shCAPS‐1, or shCAPS‐2, as detected by western blotting. (E) LN229 and GBM1 cells expressing control shRNA or shCAPS‐1 were synchronized by a double thymidine block. The levels of p‐PLK1 and t‐PLK1 were analyzed at different time points after release. (F) Pearson correlation analysis of CAPS and p‐PLK1 in 172 glioma samples. (G) Total and phosphorylated PLK1 and MYPT1 were detected by western blotting in LN229 and GBM1 cells expressing control shRNA, shCAPS, or shCAPS+MYPT1 S507E. (H) Proliferation and (I) cell cycle analysis of LN229 and GBM1 cells expressing control shRNA, shCAPS, or shCAPS+MYPT1 S507E. ***p < 0.001.
Fig 2: TRIM13 interacted with RPS27A and enhanced RPS27A ubiquitination and degradation to downregulate RPS27A expression. (a) The proteins that interacted with TRIM13 predicted by GEO database; (b) The expression levels of six predicted interacting proteins (UBA52, SPRY7, SLC25A16, RPS27A, PSENEN and KCNRG) in TRIM13 overexpressed LC cells were evaluated by western blot; (c) Exogenous protein interactions between TRIM13 and RPS27A in LC cells were detected by the Co‐IP analysis; (d) The amount of ubiquitin co‐immunoprecipitated with RPS27A in TRIM13 overexpressed LC cells was evaluated by western blot; (e, f) In the presence of CHX (2 μg/mL), the RPS27A protein levels in LC cells transfected with oe‐NC and oe‐TRIM13 was detected at 0, 2, 4, and 8 h by western blot.
Fig 3: Effects of OGD/R on the expression of RPS27A and inflammatory factors in microglia. A, Immunofluorescence to detect the localization of RPS27A and microglia in the cerebral cortical tissues of I/R-injured mice. B, Flow cytometry analysis to assess the purity of primary astrocytes/neurons (> 95%). C, RT-qPCR to detect RPS27A mRNA expression in microglia. D, ELISA to determine the expression of IFN-γ, TNF-α, IL-1β and IL-6 in microglial cells. E, MTT to measure the viability of neurons after co-culture with microglia. F, Flow cytometry to determine the apoptosis of neurons after co-culture with microglia. All cell experiments were repeated three times. ** p < 0.01, *** p < 0.001 vs. the control group. ## p < 0.01, ### p < 0.001 vs. the RPS27A-oe-NC group. && p < 0.01, &&& p < 0.001 vs. the RPS27A-sh-NC group
Fig 4: Effects of RPS27A on inflammatory factors in the brain of I/R-injured mice. A, ELISA to detect release of inflammatory factors in cerebral cortical tissues of I/R-injured mice in response to overexpression or silencing of RPS27A. B, Flow cytometry to determine immune cells in ischemic hemisphere of I/R-injured mice in response to overexpression or silencing of RPS27A. C, Immunofluorescence staining to measure number and distribution of neutrophils (indicated by red) in the whole brain of I/R-injured mice in response to overexpression or silencing of RPS27A. D, Flow cytometry to determine immune cells in peripheral blood collected from eyeballs of I/R-injured mice in response to overexpression or silencing of RPS27A. n = 6. ** p < 0.01 vs. the sham group. ## p < 0.01 vs. the RPS27A-oe-NC group. & p < 0.05, &&& p < 0.001 vs. the RPS27A-sh-NC group
Fig 5: NF‐kB signaling pathway participates in the regulation of TRIM13/RPS27A axis on LC cell proliferation, invasion and migration. (a, b) The expression levels of IKKβ and phosphorylated IKKβ (a) and NF‐kB downstream effectors including MMP9, BCL2, and caspase 3 (b) in LC cells transfected with oe‐TRIM13 and oe‐RPS27A were evaluated by western blot; (c) Cell proliferation was evaluated by CCK‐8 assay; (d, e) Cell migration (d) and invasion (e) were detected by the transwell assay.
Supplier Page from Abcam for Anti-RPS27A antibody