Anti ube2t

Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, People’s Republic of China). Protein samples were separated using 4–20% gradient SDS-PAGE, and then transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with a primary antibody at 4°C overnight followed by a horseradish peroxidase-conjugated secondary antibody for 2 hrs at room temperature. Enhanced chemiluminescence (ECL) reagents were used to visualize the bands. The following primary antibodies were used: anti-UBE2T (Proteintech, Chicago, IL, USA), anti-GAPDH (Proteintech), anti-cyclin B1 (Cell Signaling Technology, Danvers, MA, USA), anti-cyclin-dependent kinase 1 (CDK1) (Cell Signaling Technology), anti-p21 (Proteintech), and anti-p27 (Proteintech).

The cells were lysed in immunoprecipitation (IP) buffer (87787, Thermo Fisher Scientific) containing an inhibitor cocktail (4693116001, Roche). The protein concentration was measured using a BCA Kit (23225, Pierce Chemical). The protein was separated by SDS‐PAGE and then transferred to polyvinylidene fluoride membrane (Merck Millipore). The membrane was incubated with first antibody at 4°C overnight and then incubated with the second antibody for 1 h. Then the bands were observed with an enhanced chemiluminescence detection kit (36208‐A, Yeasen) and analyzed using the ChemiDoc XRS System (Bio‐Rad).
In the IP assay, the total cell protein extracts were incubated with Protein‐G magnetic beads (10004d, Invitrogen) and pre‐treated with antibody at 4°C overnight. The beads were washed with cell lysis buffer, and the immunoprecipitated samples were analyzed via immunoblotting.
The antibodies used were as follows: anti UBE2T (10105‐2‐AP), anti RPL6 (15387‐1‐AP), anti p53 (21891‐1‐AP), and anti RPL11 (16277–1‐AP) purchased from Proteintech. Anti HDM2 (Sc‐965) was purchased from Santa or Bioworld (BS1223). Anti RPL30 (A13690), anti Actin (AC026), and anti Flag (F1804) were purchased from Sigma. Anti Ub (ab7254) was purchased from Abcam.