Journal: Scientific Reports

Article Title: Doxorubicin-induced modulation of TGF-β signaling cascade in mouse fibroblasts: insights into cardiotoxicity mechanisms

doi: 10.1038/s41598-023-46216-7

Figure Lengend Snippet: TGF-β1 induced the phosphorylation of SMAD2 in both NIH3T3 cells and CFs. DOX treatments did not significantly effect SMAD2 or pSMAD2 in one hour. Western blot analysis of NIH3T3 cells ( A ) and CFs ( B ) probing SMAD2 and phosphorylated SMAD2 . Densitometry was performed on SMAD2, pSMAD2, and normalized to housekeeping protein β-actin then normalized to pSMAD2 of the group treated with TGF-β1 and without DOX. Values represent mean ± standard error (n = 4).* P -value < 0.05 compared to normalized pSMAD2 (Student’s t-test). #, P -value < 0.05 compared to treatments with DOX and with and without TGF-β1 (Student’s t-test).

Article Snippet: Primary antibodies used in this study were: recombinant rabbit anti-cellular communication network factor 2 (CCN2) monoclonal antibody diluted to a concentration of 1:1,000 (Abcam, ab209780); rabbit anti-bone morphogenetic protein 1 (BMP1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA5-103,660; RRID: AB_2852994); rabbit anti-collagen type I (COL1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA1-26204; RRID: AB_2260734); rabbit anti-suppressors of mothers against decapentaplegic homolog 2 (SMAD2) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; 51–1300; RRID: AB_2533896); rabbit anti-phosphorylated SMAD2 (pSMAD2) monoclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; MA5-15122; RRID: AB_10978317); and mouse anti-beta actin (β-actin) monoclonal antibody diluted to a concentration of 1:5,000 (Thermo Fisher Scientific; MA1-91399; RRID: AB_2273656).

Techniques: Western Blot

Journal: Frontiers in Neuroscience

Article Title: Overexpression of NT-3 in the hippocampus suppresses the early phase of the adult neurogenic process

doi: 10.3389/fnins.2023.1178555

Figure Lengend Snippet: Overexpression of neurotrophin-3 (NT-3) in the DG of the hippocampus by AAV carrying NT-3 injection. (A) Time course of the experimental procedure. (B) The expression of GFP (green) and NT-3 (red) in the DG 4–5 weeks following AAV injection. Scale bars: 100 μm. (C) Confirmation of NT-3 protein overexpression in the DG. Typical immunoblots for anti-NT-3, anti-GFP, and anti-β-actin were shown. Each lane indicates different DG samples 4–5 weeks following AAV injection. (D) The confirmation of NT-3 mRNA overexpression (left, p = 0.0012) and the effect of NT-3 overexpression on the gene expression of Trkc (right, p = 0.3282) in the ventral DG. n = 8 each for the AAV-enhanced GFP group and AAV-NT-3 group. Data are expressed using dot plots and means ± standard error of mean. ** p < 0.01. n.s., not significant.

Article Snippet: The membrane was first blocked with 5% skim milk in Tris-buffered saline for 1 h at room temperature and subsequently incubated with rabbit anti-NT-3 antibody (1:4500, kindly provided by Drs. Furuichi and Semba; ), rabbit anti-GFP monoclonal antibody (1:500, #598, Medical and biological laboratories, Japan, RRID:AB_591819), or mouse anti-β-actin monoclonal antibody (1:4000, MA5-15739, Thermo Fisher Scientific, RRID:AB_10979409) at 4°C overnight.

Techniques: Over Expression, Injection, Expressing, Western Blot