Fig 3

(A) Immunofluorescent staining of NRVM treated with vehicle, phenylephrine (100 μM) and endothelin 1 (50 nM) with anti-Ninjurin1 as primary antibody and Alexa Fluor 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 75 μm. (B) Immunoblots (left) and densitometric analysis (right) of proteins isolated from vehicle, phenylephrine (100 μM) and endothelin 1 (50 nM) treated NRVM using anti-Ninjurin1 antibody. GAPDH was used as loading control. The 16kDa (Ninjurin1-16) and 24kDa (Ninjurin1-24) Ninjurin1 isoforms are indicated. (C) Densitometric analysis of the Ninjurin1-24 signal from (B). (D, E) NRVM were transfected with control siRNA (control_siRNA) (n = 3) or siRNA targeting Ninjurin1 (Ninjurin1_siRNA) (n = 3). (D) Quantitative RT-PCR analysis of Ninjurin1 (Ninj1). GAPDH expression was used as reference. Data are presented as mean ± SD. * P < 0.05. (E) Quantitative RT-PCR analysis of Nppb and alpha-myosin heavy chain 1 (α-Mhc). GAPDH expression was used as reference. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.