Western blot

Protein samples were electrophoresed in a 10% or 12% sodium dodecyl sulfate–polyacrylamide gel and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Then, the membrane was incubated with antibodies against GDF11 (Abcam, ab124721; 1:1,000 or Biorbyt, orb101175; 1:500, which detects mature GDF11 at 12.5 kDa), Pax2 (Abcam, ab92547; 1:1,000), vimentin (CST, #5741; 1:1,000), Kim1 (R&D Systems, AF1817; 1:1,000), E-cadherin (Abcam, ab15148; 1:1,000), α-SMA (Abcam, ab21027; 1:1,000), ERK1/2 (CST, #9102; 1:1,000), p-ERK1/2 (CST, #9101; 1:1,000), cyclin D1 (Abcam, ab7958; 1:1,000), and c-Myc (Abcam, ab32072; 1:1,000) and washed with Tris-buffered saline containing 0.1% Tween 20. The membrane was incubated with the secondary immunoglobulins conjugated with horseradish peroxidase. The bound antibodies were detected with detection reagent (Pierce, Rockford, IL, USA). Quantification of the band intensity was accomplished with NIH Image J software.