Anti alb

Specific antibodies were purchased from the following commercial sources: Anti-AFP, anti-ALB, anti-CD44, anti-Evi1, anti-flag (mouse), anti-HA, anti-HNF4α, anti-H3, anti-H3K36me3, anti-Myc, anti-OCT4A, anti-P300, anti-PPM1A (rabbit), anti-pSmad2 (S465/467), anti-pSmad2 (S245/250/255), anti-pSmad3 (s423/425), anti-Smad2, anti-Smad3 (rabbit), anti-SnoN, anti-Sox2, anti-TAT, and normal rabbit IgG from Cell Signaling Technology (Danvers, MA); anti-PPM1A (mouse), anti-SC35, and anti-SETD2 were from Abcam (Cambridge, MA); Anti-Smad4 and normal mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); Anti-β-actin, and anti-flag (rabbit) from Sigma-Aldrich (St. Louis, MO); Alexa594 goat anti-mouse IgG from Life Technology (Carlsbad, CA); Dylight488 goat anti-rabbit IgG from Vector Labs (Burlingame, CA).

Acquired renal tissues were homogenized in lysis buffer (KeyGEN, Nanjing, China) containing protease inhibitors and phosphatase inhibitors. The lysates were centrifuged at 12,000 rpm for 5 min at 4°C and the supernatant was collected. The protein concentrations of mixed lysates were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of total protein were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences). After blocking, the membrane was incubated with rabbit polyclonal anti-ESR2 (1:1,000, Boster Biological Technology), anti-ESR1 (1:1,000, Santa Cruz Biotechnology), anti-AR (1:500, Cloud-Clone), anti-MAOB (1:1,000, Wuhan USCN), anti-PGR (1:1,000, Abcam), anti-GBA (1:1,000, Wuhan USCN), anti-THRB (1:1,000, Abcam), anti-ALB (1:1,000, Cell Signaling Technology), anti-CMA1 (1:1,000, Wuhan USCN) and anti-GAPDH (1:1,000, Wuhan USCN) antibodies at 4°C overnight. After washes with TBST, the membrane was incubated with a fluorescent dye-conjugated secondary anti-rabbit antibody (1:10,000, Licor) for 60 min at room temperature. The signals were visualized using the Odyssey Infrared Imaging System (Licor, NE, United States) and quantitatively analyzed by normalizing them to GAPDH levels using ImageJ software.