Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Mouse kidney, but not lung, demonstrate differences in ENaC α‐ and γ‐subunit expression with aldosterone. Lysate from mouse kidney and lung collected from animals on a high salt diet (HS) or with an aldosterone infusion (Aldo) were blotted for the presence of each ENaC subunit. (a) The Stressmarq anti‐α‐subunit antibody revealed an intense 80 kDa nonspecific band, denoted by ** and a 95 kDa full length α‐subunit, denoted by *. The top panel was exposed for a longer period (~7 min) to show the 95 kDa band and the bottom panel shows a quick initial exposure (~5 s) before saturation of the 80 kDa band occurred. (b) The blot was stripped and reprobed with a previously characterized antibody, produced in the Loffing laboratory. The panel shows two exposures, separated by a dashed line, to reveal both the full‐length 95 kDa α‐subunit, denoted by *, and a 30 kDa α‐subunit N‐terminal cleavage product. (c) The Stressmarq antibody directed against the β‐subunit shows the presence of a band at 90 kDa (denoted by *). (d) The Stressmarq antibody directed against the γ‐subunit revealed bands corresponding to a full‐length 80 kDa γ‐subunit and 70 kDa cleavage products, as indicated. (e) Signal from the kidney samples was quantified by densitometry, with each band normalized to total protein. Quantification is shown as a fold change from the average HS signal with p values shown for relationships that were significant ( p < 0.05) as assessed by multiple t ‐tests. (f) Mouse lung lysate was first probed with the Stressmarq α antibody, followed by a light chain only secondary antibody (LC‐only HRP). The blot was then stripped and reprobed again with the Stressmarq α antibody but followed by a whole IgG secondary antibody (H + L HRP). The blot was then stripped again and reprobed with the Loffing α antibody, followed by a whole IgG secondary antibody (H + L HRP). The Stressmarq antibody revealed an intense nonspecific band of ~80 kDa (denoted by **) and a full length α‐subunit migrating ~95 kDa (denoted by *). The Loffing antibody reveal both the full‐length 95 kDa α‐subunit and a 30 kDa N‐terminal α‐subunit cleavage product. Blots are representative of results from at least three separate experiments.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques: Expressing, Produced

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Immunoprecipitation eliminates the non‐specific α‐ subunit band in mouse lung tissue and FRT cells. (a) Mouse lungs were homogenized and incubated with the α‐subunit StressMarq antibody, followed by immunoprecipitation with protein G beads. Both the pulldown (left) and the lysate (right) were run together on a gel and blotted for the α‐subunit with the same StressMarq antibody as utilized for the IP. (b) Mouse kidneys collected from animals on a high salt diet (HS) or with an aldosterone infusion (Aldo) were immunoprecipitated with the StressMarq α antibody and protein G beads. Both the IP (left) and the kidney lysate (right) were run next to each other on an 8%–16% gel to probe for the α‐subunit with the Loffing α antibody. (c) Lysate from FRT cells either mock transfected or transfected with the three ENaC subunits were run for comparison (first two lanes). The lysate was incubated with either StressMarq (SM) anti‐α‐subunit antibody, beads alone, or V5‐tagged beads and subsequent pulldown was performed. The product was run on two separate gels simultaneously, with one being probed with the StressMarq anti‐α‐subunit antibody while the other was probed with an antibody directed against the HA tag. In all panels * illustrates the α‐subunit band while ** indicates to the non‐specific band. Results are representative of three separate experiments.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques: Immunoprecipitation, Incubation, Transfection

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: Subunit‐specific antibodies demonstrate linearity across a wide range of protein concentrations. Decreasing amounts of lung or kidney homogenate were probed for each subunit to determine the working range of the antibody. (a) Lung lysate ranging from 80 to 2.5 μg total protein, as denoted along the top of the blot, was probed for the α‐subunit using the StressMarq antibody. (b) The band of interest, denoted by *, was quantified and normalized to the value obtained for 40 μg (the halfway value) so that the results from three separate replicates could be combined. The dashed line demonstrates perfect linearity. (c) Kidney lysate was utilized for the β‐subunit and (e) the γ‐subunit. (d, f) The quantification of each band was again performed as described for (b), with both the full‐length and cleavage product bands being quantified for the γ‐subunit. Each graph represents results obtained from three separate experiments.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques:

Journal: Physiological Reports

Article Title: Validation of commercially available antibodies directed against subunits of the epithelial Na + channel

doi: 10.14814/phy2.15554

Figure Lengend Snippet: The α‐subunit antibody demonstrates minimal signal in AQP2‐positive cells and is mislocalized in kidney medulla. Kidney sections from mice kept on control diet (a, top) or 4 days of high K + diet (a, middle) were labeled with the StressMarq anti‐α‐subunit antibody (red on left and converted to grayscale in second column). AQP2 (green) was used as a marker of the apical lumen of principal cells. The basolateral surfaces of tubules are denoted by solid lines and the apical surface by dashed lines. A no primary control is shown for comparison (a, bottom). (b) Positive staining for the α‐subunit (red) was only observed in the inner medulla of the kidneys, with representative images displayed here. While AQP2 positive cells within the medulla (green) did show expression of ENaC (denoted by *), the majority of the signal was localized to the basolateral side of both AQP2 positive and negative tubules (shown by arrows). Scale bar represents 20 μm in all images and images are representative of three separate regions examined in three mice of each treatment.

Article Snippet: As the Stressmarq α‐subunit antibody, directed against an N‐terminal epitope, does not detect the N‐terminal 30 kDa cleavage product, we encourage companies to raise antibodies against amino acids 2–21, the antigen sequence utilized by Loffing and colleagues.

Techniques: Labeling, Marker, Staining, Expressing

Journal: PLoS ONE

Article Title: Demineralized bone matrix used for direct pulp capping in rats

doi: 10.1371/journal.pone.0172693

Figure Lengend Snippet: (A): Representative immunohistochemical images of the blank control group, the Ca(OH) 2 group and the DBM group. a: The blank control group. Pulp morphology was normal. b- f: Ca(OH) 2 group (1, 3, 7, 14, 28 days). g- k: DBM group (1, 3, 7, 14, 28 days). (B): Mean IOD value of Runx2. *means significant difference.

Article Snippet: The sections were deparaffinized with xylene, hydrated in a series of descending grades of ethanol, and then rinsed briefly with PBS for the primary antibodies; the sections were incubated overnight at 4°C with polyclonal anti- Runx2, COL I, OCN and DSP (Wuhan Boster Biological Technology, Wuhan, China).

Techniques: Immunohistochemical staining, Control

Journal: Chemical Engineering Journal

Article Title: Nanosilver-incorporated halloysite nanotubes/gelatin methacrylate hybrid hydrogel with osteoimmunomodulatory and antibacterial activity for bone regeneration

doi: 10.1016/j.cej.2019.123019

Figure Lengend Snippet: Fig. 2. Evaluation of cell viability and osteogenic differentiation in vitro. (A) MTS assay of cell culture after 1, 4, and 7 days. (B) LIVE/DEAD staining assay after 1, 4, and 7 days of cell culture (scale bar = 100 μm). (C) Osteogenic genes BMP4, RUNX2 and COL1 were assayed using RT-qPCR. (D) Osteogenic proteins detected by ELISA. *p < 0.05, **p < 0.01, NS: no statistical significance.

Article Snippet: Culture medium supernatants were collected, and the concentrations of BMP4, RUNX2 and COL1 were determined using ELISA kits (Cusabio, Wuhan, China) following the manufacturer’s instructions.

Techniques: In Vitro, MTS Assay, Cell Culture, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Chemical Engineering Journal

Article Title: Nanosilver-incorporated halloysite nanotubes/gelatin methacrylate hybrid hydrogel with osteoimmunomodulatory and antibacterial activity for bone regeneration

doi: 10.1016/j.cej.2019.123019

Figure Lengend Snippet: Fig. 4. nAg/HNTs/GelMA hybrid hydrogel enhances the osteogenic differentiation of hPDLSCs under inflammatory environment. (A) Schematic diagram showing the collection of the condition medium and hPDLSCs processing method. (B) The gene level of BMP4, RUNX2 and COL1 after osteogenic differentiation on day 14. (C) Alkaline phosphatase (ALP) staining and ALP activity after osteogenic differentiation on day 7. (D) Alizarin red staining and quantification after osteogenic dif- ferentiation on day 14. *p < 0.05, **p < 0.01, NS: no statistical significance.

Article Snippet: Culture medium supernatants were collected, and the concentrations of BMP4, RUNX2 and COL1 were determined using ELISA kits (Cusabio, Wuhan, China) following the manufacturer’s instructions.

Techniques: Staining, Activity Assay