Journal: Journal of the American Heart Association

Article Title: Cell‐Specific Actions of the Prostaglandin E‐Prostanoid Receptor 4 Attenuating Hypertension: A Dominant Role for Kidney Epithelial Cells Compared With Macrophages

doi: 10.1161/jaha.122.026581

Figure Lengend Snippet: Figure 6. The protein abundance of ENaC and NHE3 during Ang II-HTN. The proteins were extracted after 14 days of Ang II infusion. (A) Gel images of Western blotting for ENaC subtypes. (B through F) Quantification of the western blotting for ENaC subtypes. The full length or cleaved ENaCα, ENaCβ, or ENaCγ proteins in the kidney were similar in Control and KEKO mice. (G) Gel images of western blotting for NHE3 and phosphorylated NHE3 (NHE3pS552). Both NHE3 (H) and NHE3pS552 (I) protein levels were significantly reduced in KEKOs. Data are expressed as median with interquartile range and analyzed by Mann–Whitney test; **P<0.05 (effect size: large); n=6/group; ENaC indicates epithelial sodium channel; KEKO, kidney epithelial cell-specific EP4 receptor knockout; and NHE3, sodium-hydrogen antiporter 3.

Article Snippet: Mouse Immunoblot Details Antibody target ~kDa μg/lane cortex Primary ab supplier Ab host Dilution Time Secondary ab supplier Host and target Dilution Time (Pro)renin receptor Full- length cleaved ~37 ~28 40 Sigma Rb 1:1000 O/N Invitrogen GAR 680 1:5000 1 h ENaC- α Full- length cleaved ~80 ~20 40, 20 Loffing (Zurich) Rb 1:5000 1 h Invitrogen GAR 680 1:5000 1 h ENaC- β ~90 40, 20 Loffing (Zurich) Rb 1:1500 O/N Invitrogen GAR 680 1:5000 1 h ENaC- γ Full- length cleaved ~80 ~60 40, 20 Loffing (Zurich) Rb 1:1000 O/N Invitrogen GAR 680 1:5000 1 h NHE3 ~83 40, 20 McDonough Rb 1:2000 O/N Invitrogen GAR 680 1:5000 1 h NHE3pS552 ~83 5, 2.5 Santa Cruz (sc- 53 962) Mu 1:1000 2 h Invitrogen GAM 680 1:5000 1 h NKCC2 160 20, 10 DSHB (Iowa) Mu 1:6000 O/N LI- COR GAM 800 1:5000 1 h NKCC2p- S87 160 40, 20 DSTT (Dundee) Sh 1:2500 2 h Invitrogen DAS 680 1:5000 1 h Ab indicates antibody; ENAC, epithelial sodium channel; Mu, mouse; NHE3, sodium- hydrogen antiporter 3; NKCC2, Na- K- Cl cotransporter 2; O/N, overnight; Rb, rabbit; and Sh, sheep.

Techniques: Quantitative Proteomics, Western Blot, Control, MANN-WHITNEY, Knock-Out