Fig. 3

RRBP1 deficiency decreases renin transportation and secretion. A, B Representative renin immunohistochemical staining of kidneys of Rrbp1-WT and Rrbp1-KO mice. C Quantification of stain intensity in kidneys from Rrbp1-WT and Rrbp1-KO mice (scale bar = 20 μm). D Intracellular mRNA levels of RRBP1 of scramble-control and sh-RRBP1 knockdown Calu-6 cells were measured using quantitative RT-PCR. Data were analyzed using the 2-ΔΔCt method with GAPDH as the reference gene (n = 4 per group). E–H Representative immunogold staining of renin by transmission electron microscopy (TEM) in control and RRBP1 knockdown Calu-6 cells. Dark red arrow indicates the nanogold particle (scale bar = 0.5 μm). Nu, nucleus; IC, intracellular; PM, plasma membrane. I Quantification of intracellular renin particles by TEM in control and Rrbp1 knockdown Calu-6 cells. J Quantification of intracellular renin particles with a distance of more and less than 1000 nm from plasma membranes. K Protocol for collecting cell lysates and supernatant of control and RRBP1 knockdown Calu-6 cells. L Western blot analysis of RRBP1, renin protein expression in RRBP1-knockdown Calu-6 cells, and supernatant. M Protocol for forskolin-induced renin production in control and RRBP1 knockdown cells. N Western blot analysis of RRBP1, renin, and ADCY6 protein expression in RRBP1-knockdown Calu-6 cells and supernatant. Lanes 1–3 represent cells with DMSO control treatment. Lane 4–6 represents cells induced with 50 μM forskolin. WT, wild-type; KO, knock-out. Data in C, E, J were analyzed with an unpaired, two-tailed Student’s t-test; data in (D) were analyzed using a Mann–Whitney test. Data were represented as mean ± SEM. ns, no significance; *P < 0.05; ***P < 0.001