Fig 1: Western blot and its relative band intensity. (A) A representative image of protein bands of Western blotting was performed to detect the protein levels of CTLA in the treated and untreated PBMCs of celiac disease patients and healthy people and (B) the relative intensity of CTLA-4 protein expression in the mentioned treatments. To perform the western blot in each of the treated and non-treated groups, the PBMCs of 7 people with celiac disease and 7 healthy people were used. The mean band intensity obtained from each group was statistically analyzed. Error bars represent the standard deviation. One-way ANOVA was used as a statistical test. p values ≤ 0.05 were considered statistically significant. In this figure, ns means non-significant, * means p value ≤ 0.05, ** means p-value ≤ 0.01. The original image of the blotting film (uncropped) is in the supplementary section (S2).
Fig 2: Knockdown of NSUN4 suppresses exhaustion of CD8+ T cells in vitro. 50 nM NSUN4 siRNAs or scrambled siRNA was transfected into cultured CD8+NKG7+ T cells by using the PEI–PBA (polyvinylimine system modified with phenylboronic acid) system. After 72 h, cells were harvested for functional evaluation. (A) The m5C high‐throughput microarray was used to evaluate the m5C levels of DEGs in cluster 16. (B) RIP and m5C antibody‐based methylated RIP (meRIP) assays were used to verify the binding of NSUN4 with CD74 mRNA. (C) Spot hybridization and meRIP–qPCR were respectively applied to evaluate the effect of silencing NSUN4 on overall level of m5C transcripts and m5C‐modified CD74 mRNA. (D) Actinomycin D was used to incubate CD8+NKG7+ T cells the effect of silencing NSUN4 on the stability of CD74 mRNA. (E) Dual immunofluorescence staining was used to evaluate the CD7 and CD74 levels in CD8+NKG7+ T cells with or without NSUN4 knocked down. DAPI was used to mark the nucleus (blue). ×200. (F) Interference efficiencies of NSUN4 siRNAs detected with qPCR. (G) Western blotting was applied to detect the protein levels NSUN4 and CD8+ T dysfunctional markers, including PD‐1, TIM3, and CTLA‐4. (H) ELISA was used to detect the secretion of landmark immunoactive cytokines of CD8+T cells, including IFN‐γ, IL‐2, and TNF‐α. N = 3, *p < 0.05, **p < 0.01 compared with scramble.
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