Ab127574

Total protein concentration was determined by BCA protein assay kit according to the manufacturer's instructions (Thermo, MA, USA). Samples were heated at 95℃ for 10 min, and 30 mg of them was separated by 10% SDS-PAGE. Then, samples were transferred to PVDF membranes and blocked in 5% skim milk for 1 h at room temperature(RT). The membrane was incubated with the primary antibody USP49 (1:1000, ab127574, Abcam, UK), PPM1A (1:1000, ab14824, Abcam), vimentin (1:500, ab8978, Abcam), α-SMA (1:1000, ab124964, Abcam), E-cadherin (1:500, ab1416, Abcam), Smad2/3 (1:1000, ab202445, Abcam), p-Smad2/3 (1:500, ab63399, Abcam), TGF-β1 (1:1000, ab179695, Abcam), and GAPDH (1:2000, #5174, CST, MA, USA) at 4℃ overnight. Then, the membrane was incubated with secondary antibodies (A0208, A0181, and A0216, GE Healthcare/Amersham Biosciences, Piscataway, NJ, China) at RT for 1h. LAS-400 image analyzer (FujiFilm Medical Systems, CT, USA) was used to detect the HRP (GE Healthcare/Amersham Biosciences) signal.

The association between USP49 and PPM1A in HK-2 cells was assessed using Co-IP.
Briefly, Protein A/G PLUS-Agarose (sc-2003, Santa Cruz, CA, USA) was used to obtain the total protein (100μg) from cell lysis supernatant. IgG (sc-2027, Santa Cruz), anti-USP49 antibody (NBP1-81173, NOVUS, CT, USA), and antibody against PPM1A (NBP1-04333, NOVUS) were used for IP. Anti-USP49 antibody (ab127574, Abcam) and anti-PPM1A antibody (ab14824, Abcam) were used for Westernblot.An anti-ubiquitin antibody (ab7780, Abcam) was used to determine the PPM1A ubiquitination (Ub-PPM1A).