Calbindin d28k

The midbrain was dissected from the entire brain and stored at − 80 °C until the day of the experiment. Tissue was homogenized in RIPA buffer containing (in mM) 50 Tris-HCl pH 7.5, 150 NaCl, 5 MgCl₂, 1 EDTA, 1% Triton X-100, 0.25% sodium deoxycholate, 0.1% SDS, 1 sodium orthovanadate, 5 b-glycerophosphate, 5 NaF and protease inhibitor cocktail, and incubated on ice for 30 min [50 (link), 83 (link)]. The samples were centrifuged at 15,000 g for 20 min and the protein concentration of the supernatant was determined by the Bradford method.
Proteins were applied to SDS–PAGE and electroblotted on a polyvinylidene difluoride membrane. Blotting analysis was performed using a chemiluminescence detection kit. The relative levels of immunoreactivity were determined by densitometry using ImageJ.
Primary antibodies: Calbindin-D28K (1:200, Sigma Aldrich; #C9849; RRID: AB_476894); Calretinin (1:200, Millipore; #MAB1568; RRID: AB_94259); Actin (1:10000; Sigma Aldrich; #A5060; RRID: AB_476738).
Secondary antibodies: goat anti-mouse IgG (1:3000; Bio-Rad; #1706516; RRID: AB_11125547), goat anti-rabbit IgG (1:3000; Bio-Rad; #1706515; RRID: AB_2617112).
Membranes were stripped using Re-Blot Plus Strong Solution (Millipore) for 15 min at room temperature.
For each age, both genotypes were analyzed simultaneously. The different ages were analyzed in different blots.

Mice were deeply anesthetized with urethane (1.5 g/kg) and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA) as described [59 (link)]. Brains, left lateral lobes of liver, and left lungs were removed and post-fixed in 4% PFA before dehydration and embedding in paraffin. Sagittal sections from one individual mouse brain were stained with an antibody against the PC marker calbindin-D-28K (Sigma-aldrich, St. Louis, MO) using a Histostain-Plus IHC staining kit (Invitrogen; Grand Island, NY). Cells stained positive for calbindin D in the cerebellum were enumerated from 3 random 10× fields of view under an Imager M2 microscope (Zeiss; Thornwood, NY) in a blind manner, and three serial sections were scored and the average score of each mouse cerebellum was calculated. Lung and liver tissue sections were stained with anti-Ki67 antibody (Biocare Medical; Concord, CA) using a Histostain-Plus IHC staining kit (Invitrogen; Grand Island, NY). Ki67-positive cells were scored under an Imager M2 microscope (Zeiss; Thornwood, NY) in a blind manner. In lungs, Ki67-positive cells in the bronchial epithelium and alveoli were scored from 4 random 10× and 4 random 20× microscopic fields of view, respectively, and the percentage of Ki67-positive cells was calculated. In liver, Ki67 positive cells were enumerated from 4 random 10× fields of view.

Recombinant transforming growth factor beta 1 (TGF-β1) was purchased from Peprotech (Cat# 100-21, Korea). Anti-HA (hemagglutinina) agarose gel was from Sigma-aldrich (Cat# E6779, St. Louis, MO). Antibodies against phospho-Stat3 (Tyr705), total-Stat3, total-Smad2/Smad3, phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425), TGF-β, N-cadherin, vimentin, HA tag, Smad4, nitrotyrosine, Total-EGFR, phospho-EGFR (Y845, Y1068, Y1173), total-Src, and phospho-Src (Y416) were all from Cell Signaling Technology (Danvers, MA). TGFβRI, TGFβRI, AQP1 and Smad7 was from Santa Cruz (Dallas, Texas). E-cadherin and fibronectin were from BD Biosciences (Franklin Lakes, NJ). AQP2 was from Novus Biologicals (Littleton, CO, USA). THP was from AbD serotec (Kidlington, United Kingdom). Antibodies against α-SMA, calbindin D28K and β-actin were from Sigma-Aldrich (St. Louis, MO). Specific antibody against PrdxV was gifts from Dr. Ho Zoon Chae (Chonnam National University, Korea).