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:

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: This cleavage product was absent from any blots probed with the Stressmarq α‐subunit antibody despite the fact both antibodies were produced against N‐terminal epitopes and should recognize this cleavage fragment.

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: This cleavage product was absent from any blots probed with the Stressmarq α‐subunit antibody despite the fact both antibodies were produced against N‐terminal epitopes and should recognize this cleavage fragment.

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: This cleavage product was absent from any blots probed with the Stressmarq α‐subunit antibody despite the fact both antibodies were produced against N‐terminal epitopes and should recognize this cleavage fragment.

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: This cleavage product was absent from any blots probed with the Stressmarq α‐subunit antibody despite the fact both antibodies were produced against N‐terminal epitopes and should recognize this cleavage fragment.

Techniques: Labeling, Marker, Staining, Expressing

Journal: Arthritis Research & Therapy

Article Title: Pomegranate extract inhibits the interleukin-1β-induced activation of MKK-3, p38α-MAPK and transcription factor RUNX-2 in human osteoarthritis chondrocytes

doi: 10.1186/ar3166

Figure Lengend Snippet: PE inhibits IL-1β-induced signaling cascade in human OA chondrocytes . The MAPKs are a group of signaling molecules that include the p38-MAPKs which are activated by upstream MAPK kinases MKK3 and MKK6. In human OA chondrocytes, IL-1β through IL-1R activates preferentially MKK3 which leads to the activation of p38-MAPK → RUNX2 pathway. Polyphenols rich PE inhibited the IL-1β-induced activation of MKK# and p38-MAPK in human OA chondrocytes. Breaking lines indicate reduced phosphorylation. IL-1R, IL-1β receptor; MAPK, mitogen-activated protein kinase; MKK, MAPK kinases; MMPs, matrix metalloproteinases; p, phosphorylation; PE, pomegranate extract; RUNX2, runt-related transcription factor-2.

Article Snippet: Anti-MKK-3 antibody (# 32-6900), anti-MKK-6 (#32-7100) and anti-p38β antibody (#33-8700) were purchased from Zymed (San Francisco, CA, USA); anti-phospho-MKK3/6 antibody (sc-7994-R) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the anti-RUNX2 antibody (#D130-3 ) was from MBL, Naka-ku Nagoya, Japan.

Techniques: Activation Assay

Journal: Journal of cellular physiology

Article Title: Core Binding Factor β (CBFβ) is Retained in the Midbody During Cytokinesis

doi: 10.1002/jcp.24588

Figure Lengend Snippet: Subcellular localization of CBFβ during the cell cycle. A: Immunofluorescence microscopy images of SaOS-2 cells in interphase for RUNX2 (red), CBFβ (green) and DAPI (blue). RUNX2 is mainly located in the nucleus whereas CBFβ shows both nuclear and cytoplasmic localization. B: Recently divided cells show localization of CBFβ in the nucleus and cytoplasm, but also unexpectedly concentrated in the midbody. Immunofluorescence staining with Filamin A to delimitate membrane boundaries shows that the midbody is released to the extracellular space. C: A small percentage of dividing cells exhibit a DNA-containing structure (stained with DAPI) located in the midbody together with CBFβ (dotted square in overlay, enlarged on the right). D: Subcellular localization of CBFβ in SaOS-2 (left panels) and HeLa cells (right panels), during the cell cycle. β-tubulin and DAPI immunofluorescence was used to determine cell cycle stages. CBFβ is localized in the midbody during telophase.

Article Snippet: RUNX2 (8G5) mouse monoclonal (MBL International, Woburn, MA, USA), dilution used for IF (1:600).

Techniques: Immunofluorescence, Microscopy, Staining, Membrane