Fig 2: NMDA receptor expression in cultured HPASMCs. Immunofluorescence staining was performed on permeabilized HPASMCs. Network-like immunoreactive staining was observed for both GluN1 and all four GluN2 (2A–D) subunits. Control experiments with primary antibody omitted showed no immunoreactivity. Results are representative images taken from at least three separate experiments. Scale bar = 50 µM.

Fig 3: NMDA receptor mRNA expression in human pulmonary artery. RT-PCR was performed using RNA isolated from human pulmonary artery. The presence of mRNAs for GluN1 and all GluN2 subunits (2A–D) was detected in human pulmonary artery. Human brain RNA was used as a positive control.

Fig 4: Full-length NMDA receptors are expressed by cultured HPASMCs. Cultured HPASMCs were lysed and immunoprecipitated with GluN1- or GluN2- subunit specific antibodies or control non-immune IgG, and the pellets were analyzed by Western blot. Immunopositive bands containing full-length GluN1 (A) and GluN2 (2A–D) (B–E) protein were detected in immune pellets from lysates of PASMCs but not in the IgG control conditions studied in parallel. Cropped images are shown for conciseness. Full-length blots are represented in Supplementary Fig. S9. Results are representative of three separate experiments.

Fig 5: NMDA receptor expression by HPASMCs in vivo. Human lung tissue containing pulmonary artery was stained with NMDA receptor subunit specific antibodies. PECAM-1 was used as a marker for vascular endothelial cells (EC). Antibody against PECAM-1 revealed immunoreactivity within the interior surface of pulmonary artery. Immunoreactive staining for both GluN1 (B) and four GluN2 subunits (2A–D) (C–F) was observed in pulmonary artery smooth muscle cells (SMC). Control experiments with primary antibody omitted showed no immunoreactivity in either pulmonary artery endothelial cells or PASMCs (G). Scale bar = 50 µM.