FIG 4.

Regulation of IGF2R expression by vIL-6. (A) Expression of endogenous IGF2R was measured in response to increasing levels of vIL-6 (expressed from 0.1 to 0.5 μg of vector per transfected culture) relative to empty vector-transfected cells (vec, 0.5 μg). Cell extracts from cultures harvested 30 h after transfection were analyzed by immunoblotting for detection of IGF2R, vIL-6, and β-actin (loading control). Levels of IGF2R (normalized to β-actin) in vIL-6-expressing cells are shown relative to the level (set at 1.0) in empty vector-transfected cells. (B) IGF2R mRNA levels from parallel cultures transfected with 0.5 μg of empty vector or vIL-6 expression plasmid were determined by RT-qPCR, as outlined in the legend to Fig. 3B. (C) Similar analyses of IGF2R protein expression were undertaken in transfected native and VKORC1v2-null (5) HEK293T cells. The levels of IGF2R in vIL-6-expressing cells relative to levels in empty vector (vec)-transfected wild-type and VKORC1 gene-mutated cells (each set at 1.0) are shown below the IGF2R blot. Relative transfection efficiencies in WT cells (50%) and VKORC1v2-null cells (70%) were determined by cotransfection of a GFP expression plasmid. (D) VKORC1v2 (StrepII+Flag-tagged, SF) was introduced exogenously into VKORC1v2-deficient HEK293T cells by transfection of a VKORC1v2 expression plasmid (altered, codon-synonymously, in gRNA target sequences and therefore Cas9-resistant [“CR”]), along with either empty vector (vec) or vIL-6 expression plasmid to verify the role of VKORC1v2 in vIL-6-mediated augmentation of IGF2R expression. IGF2R expression levels, normalized to β-actin and expressed relative to the IGF2R levels (set at 1) in empty vector-transfected cultures, are shown below the IGF2R blot.