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: Stem Cells International

Article Title: Effects of Intermittent Administration of Parathyroid Hormone (1-34) on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

doi: 10.1155/2016/4027542

Figure Lengend Snippet: STRO-1(+) hPDLSCs exhibited strong osteogenic capacity. (a) Immunostaining for Stro-1 in isolated STRO-1(+) and STRO-1(−) hPDLSCs (magnification: 50x). The surface marker Stro-1 was stained with FITC and visualized using fluorescence microscopy. Nuclei were stained with DAPI (blue). (b) ALP activity in STRO-1(+) and STRO-1(−) hPDLSCs. (c) Osteogenic differentiated cells were stained with Alizarin red (magnification: 100x). (d) Real-time PCR and western blot analyses of Runx2 expression in STRO-1(+) and STRO-1(−) hPDLSCs. The expression of each target was normalized to that of GAPDH. Data are presented as the means ± SDs of three independent experiments performed in duplicate. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and then incubated with anti-PTH1R (1 : 100; Boster, Wuhan, Hubei, China), anti-RUNX2 (1 : 100; Boster), anti-Sp7 (1 : 250; Sigma-Aldrich), and anti- β -actin (1 : 5000; Sigma-Aldrich) antibodies overnight at 4°C.

Techniques: Immunostaining, Isolation, Marker, Staining, Fluorescence, Microscopy, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: Stem Cells International

Article Title: Effects of Intermittent Administration of Parathyroid Hormone (1-34) on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

doi: 10.1155/2016/4027542

Figure Lengend Snippet: The osteogenic capacity of STRO-1(+) hPDLSCs was enhanced by PTH. (a) Real-time PCR and western blot analyses of PTH1R, RUNX2, and SP7 expression in STRO-1(+) hPDLSCs before and after PTH treatment and induction by osteogenic-induction medium (OM). The expression of each target was normalized to that of GAPDH. (b) ALP activity in STRO-1(+) hPDLSCs. (c) Cells subjected to osteogenic differentiation were stained with Alizarin red (magnification: 100x). Data are presented as the means ± SDs of three independent experiments performed in duplicate. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and then incubated with anti-PTH1R (1 : 100; Boster, Wuhan, Hubei, China), anti-RUNX2 (1 : 100; Boster), anti-Sp7 (1 : 250; Sigma-Aldrich), and anti- β -actin (1 : 5000; Sigma-Aldrich) antibodies overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Activity Assay, Staining

Journal: Molecules

Article Title: Tetrahydroxystilbene Glucoside Regulates Proliferation, Differentiation, and OPG/RANKL/M-CSF Expression in MC3T3-E1 Cells via the PI3K/Akt Pathway

doi: 10.3390/molecules23092306

Figure Lengend Snippet: Figure 4. TSG up-regulated runt-related transcription factor-2 (Runx2), osterix (Osx), and collagen type I α1 (Col1a1) mRNA levels of the MC3T3-E1 cells. Data are represented as the mean ± SD of three determinations. * p < 0.05 and ** p < 0.01 when compared with the control group.

Article Snippet: The Runx2 antibody was purchased from Cusabio (Wuhan, China).

Techniques: Control

Journal: Molecules

Article Title: Tetrahydroxystilbene Glucoside Regulates Proliferation, Differentiation, and OPG/RANKL/M-CSF Expression in MC3T3-E1 Cells via the PI3K/Akt Pathway

doi: 10.3390/molecules23092306

Figure Lengend Snippet: Figure 8. Effect of TSG and LY-294002 on Runx2, Osx, and Col1a1. (a) mRNA and (b) Protein levels of the MC3T3-E1 cells. Data are represented as the mean ± SD of three determinations. Different letters indicate significant differences (p < 0.05).

Article Snippet: The Runx2 antibody was purchased from Cusabio (Wuhan, China).

Techniques: