Fig 1: Differentially expressed proteins screened via inflammatory cytokine array. After siRNA or pcDNA3.1 transfection for 24 h, BV2 cells were challenged with IL-4 for another 8 h to activate M2 activation, then the content of inflammatory cytokines in the supernatant fluid of cultured BV2 cells was detected by inflammatory cytokine array. Besides, the inflammatory cytokines in the lesion areas after SCI both from CD73 KO mice and WT mice were included into this detection. The differentially expressed proteins screening criteria were as follows: fluorescence intensity > 1000, fold change > 1.5, and p < 0.05. a–f Heatmaps of inflammatory cytokines fold changes and representative photographs of inflammatory cytokine array reflected the protein expression in CD73 differentially expressed cells or tissues. The differentially expressed proteins were highlighted with red boxes. g Venn diagram depicting the overlap of differentially expressed proteins identified in three groups. IL-10 and TGF-β were the intersection
Fig 2: Cartoons depicting how PrP-knockout or CG exposure may cause 5-NT overexpression, GFAP cleavage and NKA overexpression.(A) Normal cell. (B) Cell exposed to low nanomolar CG levels or engineered to be PrP deficient. (C) Cell exposed to toxic high nanomolar CG levels. Note that the cartoon depicts a generic cell and omits subtleties related to the existence of NKA paralogs and isoforms.
Fig 3: ReN VM cells respond to PrP-deficiency or cardiac glycoside exposure with an increase in the expression of a 60 kDa Coomassie-stained signal, originating from 5’-nucleotidase.(A) Observation of Coomassie-stained protein band signal at 60 kDa that is conspicuously increased in the presence of 48 hour exposure to ouabain. The blot depicting levels of actin B (ACTB) serves as an additional loading control of samples that had been adjusted for total protein levels. (B) Scheme for the on-blot digestion and mass spectrometry-based identification of the 60 kDa band-of-interest. (C) MS/MS spectrum documenting identification of 5-nucleotidase (5’-NT) as the protein underlying this band. (D) Spectral count comparison of MS-based 5’-NT identification from Western blot bands observed in the absence or presence of ouabain. (E) Western blot validation of changes to 5’-NT expression upon prolonged exposure to low levels of ouabain. (F) CRISPR/Cas9-mediated knockout of PrP mimics low levels of ouabain in its effect on steady-state 5’-NT levels. Initially, the changes in 5’-NT expression were studied in pools of CRISPR-Cas9 gene engineered ReN VM PrP-/- cells generated with two distinct gRNAs. The pools differed in the percentages of cells depleted for PrP expression. Note that the 5’-NT signal levels correlate inversely with the degree of PrP knockout (i.e., the percentage of cells exhibiting the knockout). (G) Analysis of three separate ReN VM-derived PrP-/- clones corroborates changes in 5’-NT expression relative to wild-type Ren VM cells.
Fig 4: Representative immunofluorescence images of fibroblasts and squamous cell carcinoma cells showing CD73 (green) and extracellular matrix metalloproteinase inducer (emmprin) expression (red). A, CD73 expression in ST353i cells. B, Emmprin expression in ST353i cells. C, CD73 expression in CRL‐2095 cells. D, Emmprin in CRL‐2095 cells. E, Overlay of CD73 and emmprin expression in CRL‐2095 cells. F, Overlay of CD73 and emmprin expression in ST353i cells cocultured with CRL‐2095 cells
Fig 5: Estrogen effect in CD73 expression in ovariectomized (OVX) model. (A) RT-PCR analysis of CD73 mRNA in the uterus of the OVX mice after E2 treatment for 0, 1, 6, 12, and 24 h (each group n = 4). Lactoferrin (Lf) gene was used to confirm the response of estrogen after ovariectomy. Ribosomal protein L7 (Rpl7) gene was used as an internal control. (B) Quantitative RT-PCR shows the relative fold change of CD73 expression after estrogen treatment. The relative expression value was based on the value at 0 h after E2 treatment. (C) Expression of CD73 was analyzed by Western blot analysis in the uterus of the OVX mice after E2 treatment for 0, 1, 6, 12, and 24 h (each group n = 4). β-actin was used as an internal control. (D) Relative level of CD73 protein intensity in uterus of OVX mice after E2 treatment. The relative expression value was based on the value at 0 h after E2 treatment. Data are presented as the mean intensity ± SEM.
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