2.5 Immunohistochemistry, Wheat Germ Agglutinin Staining, and Microscopy

NRVMs were grown on coverslips (pre coated with 0.2% gelatin) in 12-well plates, and treated with PE (10 μM), angiotensin II (AngII, 100 nM), PMA (2 μM), SB203580 (10 μM), or chelerythrine (10 μM) for the indicated amount of time. Following treatment, cells were fixed with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 100% methanol for 15 minutes, dehydration with 70% ethanol for 15 minutes and blocking with 6% bovine serum albumin (BSA) for 1 hour at RT. Cells were incubated in primary antibody for HuR (1:500) for 1 hour at RT followed by secondary antibody for Alexa Fluor 488 (1:2000) (ThermoFisher) for 1 hour at RT. All antibodies were made up in 0.6% BSA in PBS. For wheat germ agglutinin (WGA) staining, a Texas Red-X conjugate was used per manufacturer's instructions (ThermoFisher). All slides were imaged with the BioTek Cytation 3 image reader and quantitative assessment of HuR translocation was measured as the ratio of cytoplasmic to nuclear fluorescent intensity (adjusted for mean background fluorescence and integrated area) using ImageJ