Smad2 3

Total proteins were extracted from the kidneys or HK‐2 cells and the concentration was measured by BCA protein assay kit (Beyotime, Shanghai, China). Western blot assay was performed with the standard method. Briefly, proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). PVDF membrane (Millipore, Bedford, MA, USA) was used to electro‐transfer. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with primary antibodies: anti‐Klotho (1:500, Abcam, #203576), TGF‐β1 (1:1000, Abcam, #92486), TGFβ‐RI (1:800, Abcam, #31013), TGFβ‐RII (1:1000, Abcam #186838), α‐SMA (1:1000, CST, #56856), Collagen I (1:2000, Abcam, #34710), Collagen IV (1:1500, Abcam, #6586), Smad 2/3 (1:1000, Santa Cruz, #398844), p‐Smad 2/3 (1:800, Abcam, #63399), Smad 4 (1:1000, CST, #38454) and Smad 7 (1:1000, Santa Cruz, #365846), followed by incubation with secondary antibody at room temperature for 1 hour. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as the internal control. Protein bands were visualized by enhanced chemiluminescence (ECL) reagent and exposed using BioImaging Systems (UVP, Upland, CA, USA). The relative protein levels were quantified using the Image J software (National Institutes of Health, Montgomery, MD, USA). All the assays were performed in triplicate.

Ovaries derived from gilts at age of 30–400 days were fixed in 10% (w/v) paraformaldehyde with 0.02 MPBS (pH 7.2) at 4°C for about 2 h. Next, they were cut into vertical slices of about 0.5 cm thickness and fixed in fresh 10% (w/v) paraformaldehyde for a cumulative period of 24 h. These slices were mounted in paraffin, and serially cut into 5 μm-thick sections by Rotary Microtome (MICROM, Germany), and stained with HE. Ovarian HE sections were observed and photographed under a fluorescent microscope (Zeiss, Germany).
Immunohistochemistry detection was performed by using the anti-Rabbit HRP-DAB Cell and tissue staining kit (R&D, CTS005) and anti-Goat HRP-DAB Cell and tissue staining kit (R&D, CTS008). Immunohistofluorescence examination was performed by using TSA plus Fluorescein (PerkinElmer, NEL741001KT) and Cyanine3.5 (PerkinElmer, NEL763001KT) kit. Antibodies of BMP15 (Eterlife, EL806306-100), GDF9 (Eterlife, EL901942-100), FSHR (Eterlife, EL912710), luteinizing hormone receptor (LHR) (Eterlife, EL904141), Caspase3 (Abcam, ab13847), 3βHSD (Abcam, ab154385), p-Smad1/5 (CST, 9516), Ki67 (Abcam, ab15580) and LC3B (Arigo, ARG55799) were diluted 1:100 with PBS. Other antibodies including ALK6 (Santa Cruz, sc5679), BMPR2 (Santa Cruz, sc5683), Smad2/3 (Santa Cruz, sc8332), Smad1/5/8 (Santa Cruz, sc6031R), and p-Smad2/3 (Santa Cruz, sc11769) were diluted 1:50 in PBS.

Nuclear and cytosolic extracts or total cell lysates were prepared as described before [49 (link)]. After electrophoresis and semi-dry electroblotting onto PVDF membranes, the following primary antibodies were used for immunodetection at 1:1000-fold dilution in 5% (w/v) non-fat milk powder, 0.05% Tween20 in TBS (Tris-buffered saline; 50 mM Tris/HCl, pH 7.6, and 150 mM NaCl): Nrf2, Slug, lamin-A/C, Smad2/3, vimentin and Hsp90 (all from Santa Cruz Biotechnology, Heidelberg, Germany), E-cadherin and JNK (both from Cell Signaling, Frankfurt/a.M., Germany) or L1 (clone L1-9.3, provided by Gerd Moldenhauer, DKFZ Heidelberg). In addition, the following antibodies diluted at 1:500 in in 5% (w/v) bovine serum albumin, 0.05% Tween20 in TBS were used: phospho-JNK, phospho Smad2 (Ser465/467) and phospho Smad3 (Ser423/425) (all from Cell Signaling). After incubation overnight at 4°C, blots were exposed to the appropriate horse-radish peroxidase-conjugated secondary antibody (Santa Cruz) diluted (1:1000) in blocking buffer and developed using the Dura detection kit (Perbio Sciences, Bonn, Germany). Data acquisition was done with the Chemidoc-XRS gel documentation system (BioRad, Munich, Germany) using the Quantity One software (Bio-Rad). Hsp90 and lamin-A/C served as loading control for total cell lysates and nuclear extracts, respectively.