Figure 5. GPR126 modulates LRP1 expression levels.

(A–C) RNA sequencing of fBECs from WT and Gpr126^iECKO mice at P18. (A) Normalized enrichment scores from GSEA for differentially expressed genes in Gpr126^iECKO (P ≤ 0.01, adjusted P ≤ 0.05, at least 30 altered genes). (B) Heatmap displaying z scores of leading-edge genes from GSEA for selected GO terms (n = 4 WT, n = 4 Gpr126^iECKO mice per replicate). (C) Log₂ fold change of selected genes differentially expressed in Gpr126^iECKO. (D) Real-time qPCR of Lrp1 in fBECs (n = 7 WT, n = 9 Gpr126^iECKO). (E and F) Immunoblotting for LRP1 in fBECs from WT and Gpr126^iECKO mice at P18 (E) normalized over GAPDH (F) (n = 3 WT, n = 3 Gpr126^iECKO). (G) FISH confocal images for Lrp1 (red) and Cldn5 (green) mRNA in cBECs from WT and Gpr126^iECKO mice at P18. Scale bar: 20 μm. (H) Single-molecule RNA (smRNA) of Lrp1 per nucleus in G. Each symbol represents a field of 40 cells (n = 9 WT, n = 6 Gpr126^iECKO). (I and J) Immunoblotting for LRP1 (I) normalized over vinculin (J) in cBECs transfected with control or Gpr126 siRNA and treated with collagen IV or PBS (I) (n = 6 WT mice). (K and L) Immunoblotting for total CREB and phospho-CREB S133 (K) normalized over vinculin (L) in cBECs treated as in I (n = 12 WT mice). (M–O) Immunoblotting for LRP1 and GPR126 in cBECs from adult WT and Lrp1^iECKO (M), normalized over vinculin (N and O) (n = 3 WT, n = 3 Lrp1^iECKO). (P) GPR126 regulates LRP1 expression and vice versa. 1. Collagen IV–GPR126 interactions induce cAMP, phospho-CREB, and LRP1. 2. LRP1 localizes at the plasma membrane. 3. This supports GPR126 expression and signaling. Dashed arrow, undefined LRP1-mediated induction of GPR126. Data are shown as means ± SD. (F, J, K, N, and O) Each symbol represents an experiment. (D, F, H, N, and O) Unpaired t tests with Welch’s correction; (J and L) Brown-Forsythe and Welch’s ANOVA, Dunnett’s T3 multiple-comparison tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.