Journal: Frontiers in endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children.

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: FIGURE 4 Local effects of TNF on humanin, PCNA, SOX9 and TRAF2 expressions in growth plates tissue specimens (n=3) in patient 1 and patient 2 analyzed separately. Quantitative analysis of (A, E) humanin staining, (B, F) PCNA staining, (C, G) SOX9 staining, and (D, H) TRAF2 staining. Error bars indicate mean ± SE. Students t-test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining

Journal: Frontiers in endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children.

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: FIGURE 5 TNF suppressed SOX9, PCNA and TRAF2 expressions in human growth plate tissue specimens (n=6) obtained from 2 children or human chondrocytes. (A, B) Quantitative analysis of SOX9 staining (yellow arrows), calculated as number of positive cells per mm². (C) Relative expression of SOX9 assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (D, E) Quantitative analysis of PCNA staining (yellow arrows), calculated as number of positive cells per mm². (F) Relative expression of PCNA assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (G) Western blot analysis of SOX9 and PCNA expressions in HCS- 2/8 cells treated with TNF (100 ng/ml). (H, I) Quantification of SOX9 and PCNA expressions by three independent Western blot experiments. (J, K) Quantitative analysis of TRAF2 staining (yellow arrows), calculated as number of positive cells per mm². Error bars indicate mean ± SE. Students t- test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

doi: 10.1074/jbc.m114.587808

Figure Lengend Snippet: FIGURE 2. The NIK IBM is important for reinforcing the interactions within the c-IAPTRAF2TRAF3NIK complex and is necessary for proper E3 ubiquitin ligase targeting of NIK by c-IAP1. A, HEK293T cells were transfected with TRAF3 siRNA to suppress endogenous TRAF3 expression along with plasmids encoding NIK-GFP or NIK (A2G/V3G)-GFP and FLAG-cIAP1 or FLAG-cIAP1 (H588A). Cell lysates were prepared after 48 h of incubation and analyzed by Western blot using anti-GFP, anti-TRAF3, and anti-FLAG-HRP. B, FLAG-cIAP1 constructs with either NIK-GFP or NIK (A2G/V3G)-GFP were transfected in HEK293T cells, and co-IPs were conducted using an anti-NIK antibody. Eluates were immunoblotted by anti-FLAG-HRP for detection of the c-IAP1-NIK interaction. In addition, the levels of endogenous TRAF2 and TRAF3 in the c-IAPTRAFNIK complex were also analyzed by Western blot. The expression levels of NIK, cIAP1, TRAF2, and TRAF3 in input lysates were confirmed by immunoblotting with indicated antibodies. CON, control; EV, empty vector.

Article Snippet: Antibodies used for immunoblotting: NIK (H-248), TRAF2 (C-20), TRAF3 (G-6), GST (Z-5) (Santa Cruz Biotechnologies); TRAF2 (BD Pharmingen); FLAG-HRP, HA-HRP, FLAG M2, and -actin (Sigma); GFP (Novus).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Incubation, Western Blot, Construct, Control, Plasmid Preparation