PMC

Protein levels were detected by Western blot using 8H9 or MAB1027. Actin levels served as loading control.

(A) Either 0 ug (− 4Ig-B7-H3) or 3 ug (+ 4Ig-B7-H3) recombinant human 4Ig-B7-H3 was used for blocking. Natively expressed B7-H3 was detected by 8H9, while L1-CAM by 3E7. Representative FACS histograms with mean fluorescence intensity (MFI) from three independent experiments were shown. A non-specific MoAb TIB114 served as the staining control. (B) Various amounts of recombinant human 4Ig-B7-H3 (as indicated) were used for blocking. Bar graphs with MFI relative to 0 ug 4Ig-B7-H3 were shown. Mean + SD.

miR-29 (a, b, c) levels were measured by qRT-PCR. RNU48 level served as the endogenous control, and miR-29 levels were normalized to the mean of 18 normal tissues (A): 18 normal tissues (detailed in the Results), 40 neuroblastomas, 8 sarcomas, 8 brain tumors, and 8 8H9+ (positive) tumor cell lines. (B) normal CNS tissues were normalized to liver.

mRNA levels were measured by qRT-PCR. Geometrical mean of HPRT1 and SDHA transcript levels served as the endogenous control, and expression levels in log scale were fold change relative to peripheral blood mononuclear cells (PBMC); number of samples for each histological type detailed in Results, and normal liver tissue (8H9 positive) was singled out for direct comparison.

(A) Western blot detection of 8H9 antigen using 8H9. The highly glycosylated 8H9 antigen migrated at ∼ 90 KD on 4-15% Tris-Glycine SDS PAGE under nonreducing conditions. LAN1 - 8H9 positive cell line; Daudi – 8H9 negative cell line; whole cell lysates (WCL), nuclei fraction (N), cytosolic fraction (C), and membrane fraction (M). The purification procedure was monitored by (B) 8H9 Western blot using Invitrogen SeeBlue Plus2 Pre-Stained Standard as the protein molecular weight marker and (C) silver staining. Fractions designation detailed in Methods. Amount of aliquots loaded (ratio to total amount) were marked at the bottom of the panels. (D) Confirmation of 8H9 antigen as 4Ig-B7-H3 by Western blot using both 8H9 and MAB1027.

(A) Alignment of miR-29a with the target site derived from B7-H3 3′UTR. Note the seed complementarity at the 5′ end of miR-29a (bolded). A single-base mutant (underlined) was also synthesized. (B) Luciferase activity in HeLa cells transiently cotransfected with firefly luciferase constructs and miRNA mimics. Luciferase vectors were parental (Control), with the B7-H3 3′UTR insert (B7-H3-WT), or with the mutant insert (B7-H3-mt). miRNA mimics were miR-29a or NC (negative control). Luciferase values were normalized to Control independently for NC and miR-29a because each of these miRNAs differentially influenced the values of the renilla luciferase transfection control. Mean + SD. (C) B7-H3 expression in NB1691 cells transiently transfected with miRNA mimics, inhibitors, or mimics plus inhibitors. miRNAs were miR-29a or NC (negative control). Protein expression was measured by 8H9 immunofluorescence staining, and analyzed by FACS. Representative FACS histograms with mean fluorescence intensity (MFI) from three independent experiments were shown, and SD of the mean of the triplicates was < 5%. A non-specific MoAb TIB114 served as the staining control.