ps552 nhe3  (Santa Cruz Biotechnology)

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

Journal: European journal of pharmacology

Article Title: Eukaryotic elongation factor 2 kinase inhibitor, A484954 inhibits perivascular sympathetic nerve stimulation-induced vasoconstriction in isolated renal artery.

doi: 10.1016/j.ejphar.2022.175042

Figure Lengend Snippet: Fig. 6. Expression (a) and dimerization (b) of neuronal nitric oxide synthase (nNOS), and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at Ser239 (c) and Src (d) in E (+) Wistar rat isolated renal artery in the presence of TNS stimulation (60 V, 50 Hz, 30 s) without (vehicle) or with A484954 (10 μM, 10 min) as determined by Western blotting. Specifically, the expression of nNOS (dimer and monomer froms) was determined by Western blotting under non-reducing condition by using total nNOS antibody (b). The expression of nNOS (a) and phosphorylation of Src (p-Src) (d) were normalized to total-actin (t-actin) expression, and phosphorylation of VASP (p-VASP) was normalized to total-VASP (t-VASP) expression (c). The results shown as fold increase relative to vehicle were expressed as mean ± SEM (a: N = 6, b: N = 4, c: N = 4, d: N = 6). N represents the number of rats.

Article Snippet: Antibody sources were as follows: phospho-eEF2 Thr56 (No. 905- 775-100) (Assay designs, Ann Arbor, MI, USA), total-eEF2 (No. A301688A-T) (Bethyl Laboratories, Montgomery, TX, USA), neuronal NO synthase (nNOS) (No. sc-53962) (Santa Cruz Biotechnology, Dallas, TX, USA), phospho-vasodilator-stimulated phosphoprotein (VASP) Ser239 (No. ab194747) (Abcam, Cambridge, UK), total-VASP (No. sc-46668) (Santa Cruz Biotechnology), phospho-Src Tyr416 (No. 6943P) (Cell signaling Technology, Beverly, MA, USA), and total-actin (No. MAB1501) (Sigma-Aldrich).

Techniques: Expressing, Phospho-proteomics, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: NFAT5 Is Involved in GRP-Enhanced Secretion of GLP-1 by Sodium

doi: 10.3390/ijms22083951

Figure Lengend Snippet: Effect of gastrin and GLP-1 on sodium transporter/pump/exchanger in human renal proximal tubule cells (hRPTCs). The hRPTCs were treated with gastrin (100 nM), GLP-1 (10 nM), or gastrin plus GLP-1 for 3 h. ( A ) SGLT2 protein, quantified by immunoblotting, was normalized by tubulin n = 3/group. * p < 0.05 vs. VEH (vehicle)- or gastrin-treated group. ( B ) NKA (Na + -K + /ATPase) protein, quantified by immunoblotting, was normalized by tubulin. n = 3/group. * p < 0.05 vs. VEH or GLP-1 group. # p < 0.05 vs. gastrin-treated group. ( C ) Total NHE3 protein, quantified by immunoblotting, was normalized by tubulin. n = 3/group, no differences among the groups. ( D ) Immunoblots of phospho-NHE3. 1 set of blots from three independent experiments is shown. ( E ) Phospho-NHE3 (SEs of VEH are too small to show), quantified by immunoblotting, was normalized by GAPDH. n = 3/group. * p < 0.05 vs. VEH group. # p < 0.05 vs. others. One-way ANOVA, Holm–Sidak test.

Article Snippet: The primary antibodies for renal sodium transport were: rabbit polyclonal SGLT2 antibodies (1:1000, ab37296; Abcam, Cambridge, MA, USA); rabbit polyclonal NHE3 antibodies (1:1000, MP635E; GenWay BioTech Inc., San Diego, CA, USA), mouse monoclonal Na + -K + /ATPase antibodies (1:1000, 05-369; Millipore Sigma, Burlington, MA, USA); and mouse monoclonal p-serine552-NHE3 and p-serine605-NHE3 antibodies (1:200, sc-53962 and sc-53961, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot