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. 2024 Jul 25;27(9):110587. doi: 10.1016/j.isci.2024.110587

Figure 1.

Figure 1

Expression and biochemical characterization of B7-H5 in renal cells

(A) Boxplot of B7-H5/VISTA mRNA expression in ccRCC tumor tissue (shown in red) in comparison to normal tissue (in gray). Data are represented in a logarithmic scale (Log2) and obtained from 523 tumor samples and 72 normal tissue samples (data from TCGA). The asterisk indicates a statistically significant difference (p < 0.01).

(B) Western blot of total lysates from HEK293 cells ectopically expressing B7-H5 Flag. Cells were transfected with pcDNA3.1+/C-DYK empty vector (EV) or containing B7-H5 Flag, and a Western blot was performed using anti-Flag or anti-GAPDH (loading control) antibodies. Migration of molecular weight markers is shown.

(C) Immunofluorescence confocal microscopy images at 63X showing subcellular localization of ectopically expressed B7-H5 Flag in HEK293 and COS-7 cells. Anti-Flag primary antibody and Alexa Fluor 488-conjugated secondary antibody were used for visualization of B7-H5 Flag (in green). Nuclei were stained with DAPI (in blue).

(D) Western blot of total lysates from HEK293 cells ectopically expressing B7-H5 Flag. Cells were transfected with pcDNA3.1+/C-DYK empty vector (EV) or containing B7-H5 Flag. Lysates were kept untreated or processed for PNGase F protocol in the absence (−) or in the presence of PNGase F enzyme (+). Western blot was performed using anti-Flag, anti-B7-H5, or anti-GAPDH antibodies. Migration of molecular weight markers is shown.

(E) Western blot of total lysates from HEK293 cells ectopically expressing B7-H5 Flag or B7-H5. Cells were transfected with pcDNA3.1+/C-DYK empty vector (EV), or containing B7-H5 Flag or B7-H5. Western blot was performed using anti-Flag, anti-B7-H5, or anti-GAPDH antibodies. Migration of molecular weight markers is shown.

(F) Western blot of total lysates from HEK293 cells ectopically expressing B7-H5.Lysates were kept untreated or treated with PNGase F, as indicated. Anti-B7-H5 was used for identification of B7-H5 protein and anti-GAPDH was used as a loading control.