rabbit polyclonal anti nhe3  (Proteintech)

Journal: Frontiers in Physiology

Article Title: Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel

doi: 10.3389/fphys.2021.787521

Figure Lengend Snippet: Characterizing the CD-principal-cell targeted mTOR deletion. (A) To evaluate cellular location of Cre-recombinase activity, LacZ reporter mice were crossed with Aqp2 c re transgenic mice and the kidneys of offspring probed for β-galactosidase activity- blue stain; left (Cre-negative); right (Cre-positive) demonstrating staining specific to renal collecting duct in Cre positive mice. mTOR CD-KO mice were generating by crossing male Mtor t m1.2K oz /J with female Aqp2 c re mice. Western blots of cortex and inner medulla homogenates (equal amounts of protein were loaded in each lane, 10 μg/lane) from wild-type (WT) and knockout (KO) (B) male and (C) female mice ( n = 6/group) probed with anti-mTOR polyclonal antibody; (D) densitometric summaries of western blotting of mTOR in cortex (CTXH) and inner medullary homogenates (IMH, mean ± sem); (E) dot blot of inner medulla (IMH), outer medulla (OMH), and cortex (CTXH) homogenates (2 μl/dot) from WT and KO male and female mice ( n = 6/group) probed with anti-mTOR antibody. (F) Ponceau S staining of proteins on dot blot for loading correction of mTOR dot blot; (G) density summary of dots normalized by Ponceau intensity. Two-way ANOVA (genotype × sex) is shown below bars; * indicates a significant ( p < 0.05) difference between WT and KO.

Article Snippet: The rabbit polyclonal NHE3 antibody was from StressMarq Biosciences (catalog #SPC-400).

Techniques: Activity Assay, Transgenic Assay, Staining, Western Blot, Knock-Out, Dot Blot

Journal: Frontiers in Physiology

Article Title: Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel

doi: 10.3389/fphys.2021.787521

Figure Lengend Snippet: Epithelial sodium channel (ENaC) activity and expression in kidney is reduced in KO mice. (A) Natriuretic sensitivity to the ENaC-select antagonist, benzamil was used as a surrogate for in vivo ENaC activity. Male mice ( n = 6/genotype) were given a single intraperitoneal injection of benzamil @ 1.4 mg/kg⋅bw, urine collected in the subsequent 4-h period then measured for volume and Na+. The experiment was repeated the following week with 4.3 mg/kg⋅bw dose. (B) Male mice were fed NS or LS diet for 7-days ( n = 6/genotype/diet) then euthanized and kidneys harvested. (B) Western blots of kidney cortex homogenates loaded with 30 μg/lane were probed with rabbit polyclonal antibodies against α-, β-, or γ-ENaC as indicated. A reprobe with β-actin (representative image) was used to normalize bands. (C) Band density summary (mean ± sem) and results of 2-way ANOVA (genotype × treatment). The major bands for all three subunits were significantly reduced ( p < 0.05) in the KO, relative to WT. The LS diet also significantly affected abundance of all bands analyzed.

Article Snippet: The rabbit polyclonal NHE3 antibody was from StressMarq Biosciences (catalog #SPC-400).

Techniques: Activity Assay, Expressing, In Vivo, Injection, Western Blot

Journal: Frontiers in Physiology

Article Title: Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel

doi: 10.3389/fphys.2021.787521

Figure Lengend Snippet: Abundances of non-collecting-duct sodium reabsorptive proteins. Western blots of kidney (A) cortex homogenates from NS and LS diet fed male WT and KO mice were probed with antibodies against candidate proteins. (B) Band density (mean ± sem) summary ( n = 6 mice/treatment/genotype) and two-way ANOVA p-values for major bands. NHE3, sodium hydrogen exchanger, type 3; NaPi-2, sodium phosphate cotransporter, type 2; NBCe1, electrogenic sodium bicarbonate cotransporter, type 1; NKCC2, bumetanide-sensitive sodium potassium 2 chloride cotransporter, type 2; NCC, thiazide-sensitive sodium chloride cotransporter. KO mice had increased protein levels of NaPi-2 under LS diet, NBCe1 under NS diet, and NCC under both diets relative to WT.

Article Snippet: The rabbit polyclonal NHE3 antibody was from StressMarq Biosciences (catalog #SPC-400).

Techniques: Western Blot

Journal: Frontiers in Physiology

Article Title: Long-Term Angiotensin II Infusion Induces Oxidative and Endoplasmic Reticulum Stress and Modulates Na + Transporters Through the Nephron

doi: 10.3389/fphys.2021.642752

Figure Lengend Snippet: Protein expression by western blotting and by immunofluorescence (Arbitrary Units).

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit monoclonal anti-PERK [1:3000, #3192 (C33E10), Cell Signaling, reactivity H, M, and R]; rabbit monoclonal anti-phospho-Thr980 PERK (1:5000, #G.305.4, Thermo Fisher, Rockford, United States, reactivity M and R); rabbit monoclonal anti- eIF2α [1:1000, #5324S (D7D3), Cell Signaling, reactivity H, M, R, and Mk]; rabbit monoclonal anti-phospho Ser51 eIF2α [1:1000, #3597 (119A11), Cell Signaling, reactivity H, M, R, Mk, and Dm]; rabbit polyclonal anti-α-SMA (1:1500, #ab5694, Abcam, Cambridge, United Kingdom, reactivity H and M); rabbit polyclonal anti-NHE3 (1:1000, #SPC400D, StressMarq, Victoria, BC, Canada, reactivity H and R); mouse monoclonal anti-phospho-Ser552 NHE3 Clone 14D5 [1:3000, #MABN2415, (Sigma Aldrich, St. Louis, MO, United States), reactivity M, H, and R]; rabbit polyclonal anti-NCC (1:5000, #GTX41969, GeneTex, Irvine, CA, United States, reactivity H, M, and R); rabbit polyclonal anti-NKCC2 (1:1000, #38436, Cell Signaling, reactivity H, M, and R); rabbit polyclonal anti-NHE1 (1:2000, #ab67313, Abcam, reactivity R and H) and rabbit polyclonal anti-ENaC β (1:1000, SPC-404D, reactivity H, M, and R).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Frontiers in Physiology

Article Title: Long-Term Angiotensin II Infusion Induces Oxidative and Endoplasmic Reticulum Stress and Modulates Na + Transporters Through the Nephron

doi: 10.3389/fphys.2021.642752

Figure Lengend Snippet: Effect of Ang II and/or losartan on the protein expression levels of kidney Na + transporters. Protein expression in kidney cortex tissue from sham rats and the rats treated with Ang II and/or losartan. Representative immunoblots showing total Na + /H + exchanger isoform 3 (NHE3), phospho/S552/NHE3, Na + /H + exchanger isoform 1 (NHE1) and epithelial Na + channel subunit beta (ENaCβ; A ). For immunoblot analysis, 50 μg samples of protein were resolved by 10% SDS-PAGE. The values are expressed as the mean ± SD ( n = 5–8 per group), and β-actin was used as a loading control. The quantification of bands is represented as total NHE3 (B) , phospho-S552 NHE3 (C) , NHE1 (D) , and ENaC β (E) . Sham (control) group; AII, angiotensin II, Los, losartan; and AII/Los, angiotensin II/losartan. + p < 0.05, ++ p < 0.01, and +++ p < 0.001 for the AII/Los interaction, as indicated by two-way ANOVA. * p < 0.0, ** p < 0.01, and *** p < 0.001 for AII vs sham or # p < 0.05, ## p < 0.01, and ### p < 0.001 for AII/Los vs AII, as indicated by multiple comparisons.

Article Snippet: The membranes were incubated with the following primary antibodies: rabbit monoclonal anti-PERK [1:3000, #3192 (C33E10), Cell Signaling, reactivity H, M, and R]; rabbit monoclonal anti-phospho-Thr980 PERK (1:5000, #G.305.4, Thermo Fisher, Rockford, United States, reactivity M and R); rabbit monoclonal anti- eIF2α [1:1000, #5324S (D7D3), Cell Signaling, reactivity H, M, R, and Mk]; rabbit monoclonal anti-phospho Ser51 eIF2α [1:1000, #3597 (119A11), Cell Signaling, reactivity H, M, R, Mk, and Dm]; rabbit polyclonal anti-α-SMA (1:1500, #ab5694, Abcam, Cambridge, United Kingdom, reactivity H and M); rabbit polyclonal anti-NHE3 (1:1000, #SPC400D, StressMarq, Victoria, BC, Canada, reactivity H and R); mouse monoclonal anti-phospho-Ser552 NHE3 Clone 14D5 [1:3000, #MABN2415, (Sigma Aldrich, St. Louis, MO, United States), reactivity M, H, and R]; rabbit polyclonal anti-NCC (1:5000, #GTX41969, GeneTex, Irvine, CA, United States, reactivity H, M, and R); rabbit polyclonal anti-NKCC2 (1:1000, #38436, Cell Signaling, reactivity H, M, and R); rabbit polyclonal anti-NHE1 (1:2000, #ab67313, Abcam, reactivity R and H) and rabbit polyclonal anti-ENaC β (1:1000, SPC-404D, reactivity H, M, and R).

Techniques: Expressing, Western Blot, SDS Page