Journal: American Journal of Physiology - Renal Physiology

Article Title: Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

doi: 10.1152/ajprenal.00304.2011

Figure Lengend Snippet: Characterization of a novel anti-α-epithelial sodium channel (ENaC) antibody. A: to test the specificity of the affinity-purified antibody, Fisher rat thyroid (FRT) cells were transiently transfected with the following constructs: green fluorescent protein (GFP) alone, GFP-tagged mouse α-ENaC, untagged mouse β,γ-ENaC only, and hemagglutinin (HA)-tagged mouse α-ENaC with untagged β,γ-ENaC. Whole cell lysates from these samples were loaded onto gels, and proteins were resolved by SDS-PAGE. Blots were probed with i) the α-ENaC antibody (1:1,000), ii) anti-HA antibody (1:1,000), iii) the α-ENaC antibody preincubated with the immunizing peptide (1 μg/μl), and iv) anti-β actin (1:1,000) as a loading control. From the detected bands, it is clear that the antibody recognizes the full-length α-ENaC and not β- or γ-ENaC. The addition of a GFP tag shifted the apparent weight up by ∼20 kDa as expected, and use of the anti-HA antibody produced a band at the same weight, indicating that the antibody is recognizing α-ENaC specifically. In addition, preincubation with the immunizing peptide eliminated the specific signal. It is therefore likely that this antibody is specific for α-ENaC in the mpkCCD cells. B: to demonstrate the specificity of fluorescent immunolabeling, mpkCCD cells cultured on filters were fixed (as described in materials and methods) and permeabilized, and indirect immunofluorescent labeling was performed using an Alex 568 anti-rabbit secondary antibody (Invitrogen) at 1:2,000 dilution with the following conditions: i) α-ENaC as the primary antibody, ii) no primary antibody (to test for nonspecific secondary labeling), and iii) α-ENaC antibody preincubated with immunizing peptide (as for the Western blots in A). Bars = 10 μm.

Article Snippet: Other antibodies used included anti-Rab11a (BD Biosciences, San Jose, CA), anti-GFP (Abcam, Cambridge, MA), anti-actin (Sigma), monoclonal anti-β-ENaC (Santa Cruz Biotechnology, Santa Cruz, CA), anti-γ-ENaC antibody (StressMarq, Victoria, BC), and fluorescently tagged phalloidin-Alexa 633 (Invitrogen, Carlsbad, CA).

Techniques: Affinity Purification, Transfection, Construct, SDS Page, Produced, Immunolabeling, Cell Culture, Labeling, Western Blot

Journal: Frontiers in Physiology

Article Title: Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats

doi: 10.3389/fphys.2021.656460

Figure Lengend Snippet: Effect of dual versus single RAS blockade on the expression of total and cleaved ENaC subunits in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (75 μg for α-ENaC, β-ENaC, and γ-ENaC and 20 μg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) Full-length (FL) α-ENaC, (B) cleaved α-ENaC, (C) β-ENaC, (D) cleaved γ-ENaC. The values represent individual measurements and the means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs. 2K; ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. 2K-1C; &&& P < 0.001 and &&&& P < 0.0001 vs. 2K-1C + E; $$ P < 0.01 and $$$$ P < 0.0001 vs. 2K-1C + L.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% non-fat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and the following specific primary antibodies overnight (4°C): polyclonal antibody against podocin (1:1,000; Santa Cruz Biotech, sc-21009), polyclonal antibody against nephrin (1:1,000; Abcam, Cambridge, MA, Ab 58968), polyclonal antibody against megalin (1:50,000; a gift from Dr. Daniel Biemesderfer) , polyclonal antibody against cubilin (1:1,000; Santa Cruz Biotech, sc-20609), polyclonal antibody against the ClC-5, H + /Cl – exchange transporter 5 (1:1,000; Alpha Diagnostic International, CLC51-A, San Antonio, TX, United States) , polyclonal antibody against α-ENaC (1:1,000; Alomone Labs, ASC-030, Jerusalem, Israel), polyclonal antibody against β-ENaC (1:1,000; StressMarq Biosciences, SPC-404, Victoria, British Columbia, Canada), polyclonal antibody against cleaved γ-ENaC (1:1,000; Alomone Labs, ASC-011), or actin (1:5,000; Abcam ab179467).

Techniques: Expressing, Isolation, SDS Page, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Estrogen Impairs Adipose Tissue Expansion and Cardiometabolic Profile in Obese-Diabetic Female Rats

doi: 10.3390/ijms222413573

Figure Lengend Snippet: Ovariectomy and E2 treatment effects on adiponectin and mediators of adiponectin signaling pathway in female ZDF rats. ( A ) Serum adiponectin levels. WAT protein levels of ( B ) adiponectin, ( C ) ADIPOR1 and ADIPOR2, ( D ) APPL1, ( E ) PPARG. ADIPOR, adiponectin receptor protein; APPL1, adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1; PPARG, peroxisome proliferator activated receptor gamma. Protein bands were normalized to loading control GAPDH protein intensity and expressed in arbitrary units (au) related to LEAN group. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t -test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test.

Article Snippet: Antibody for adiponectin (26 kDa, 3553) was supplied by ProSci (Poway, CA, USA); antibodies for ADIPOR1 (43 kDa, sc-99183), ADIPOR2 (50 kDa, sc-46755), GAPDH (37 kDa, sc-365062), GLUT4 (53 kDa, sc-7938) and PPARG (53 kDa, sc-7196) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies for AKT (60 kDa, cs-2920), pAKT (60 kDa, cs-4060) and APPL1 (82 kDa, cs-3858) were supplied by Cell Signaling (Danvers, MA, USA).

Techniques: