Soluble Corticotropin-Releasing Hormone Receptor 2 Splice Variant Is Efficiently Translated But Not Trafficked for Secretion

Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Materials and Methods - Supplemental Materials and Methods
• Supplemental Table 1 - Primers
• Supplemental Figure 1 - sCRH-R2α coexpression does not alter CRH-R2α or CRH-R1 subcellular localization, or vice versa. Cos-1 cells were transfected with CRH-R2αV5 or CRH-R1V5, plus sCRH-R2α or empty vector, and processed for immunofluorescence as described in supplemental methods. Each row represents a single field of cells, transfected as indicated on the left and sequentially recorded for DAPI (blue, 1st column), anti-sCRH-R2α signal (green, 2nd column), and anti-V5 signal (red, 3rd column). Merged images are shown for each row in the 4th column. CRH-R2αV5 and CRH-R1V5 are expressed on the membrane and in secretory organelles, and localization is unaltered by coexpression of sCRH-R2α. Likewise, sCRH-R2α localization is unchanged by CRH-R1 or CRH-R2α coexpression and remains in the cytoplasm and nucleus (Compare to Fig.5). All cells were treated with 20μM MG132 for 3 hrs prior to processing to increase sCRH-R2α protein levels; however, this did not affect localization. Scale bars in each DAPI panel represent approximately 20μm.
• Supplemental Figure 2 - sCRH-R2α coexpression does not alter Ucn I-induced cAMP signaling though CRH-R2α or CRH-R1. HEK293 cells co-transfected with CRH-R2α or CRH-R1 and sCRH-R2α or empty vector at equimolar ratios were treated with 100nM Ucn I for 15 min, and cellular cAMP was measured. Bars show the average cAMP levels (normalized for transfection efficiency and expressed relative to the largest value) ± SEM of 3 independent replicates for CRH-R2α- or CRH-R1 cells co-transfected with empty vector (black bars) or sCRH-R2α (gray bars). Student’s T-test was used to confirm the lack of significant difference between empty vector and sCRH-R2α transfected samples for each condition (n = 3, P > 0.5).