Protease inhibitors cocktail and phosphatase inhibitors

Cells were lysed with cell lysis buffer (Cell Signaling Technology, Boston, USA, #9803S) containing protease inhibitors cocktail and phosphatase inhibitors (Bimake, shanghai, China). For Co‐IP, after centrifugation, supernatants were collected and incubated with appropriate antibodies for 1 h at 4 °C, followed by protein G beads (Santa Cruz Biotechnology, CA, USA, #sc‐2002) overnight. After incubation, beads were washed with IP buffer. Immunoblot assays were performed with specific antibodies. The following antibodies were used for Co‐IP or immunoblot assay: DKC1 (Santa Cruz Biotechnology, CA, USA, #sc‐373956), RPL10A (Proteintech, Chicago, USA, #16681‐1‐AP), RPL22L1 (Immunoway, TX, USA, #YN1235), RPL34 (Affinity Biosciences, Cincinnati, OH, USA, #DF3708), RPS3 (Cell Signaling Technology, Boston, USA, #9538), Myc (RM1003, Ray Antibody, Beijing, China), Flag (Sigma, St. Louis, USA, #F1804), and His (Proteintech, Chicago, USA, #66005‐1‐Ig).

Cells or tissues were lysed with cell lysis buffer (50 × 10⁻³m Tris–HCl PH 7.5, 150  × 10⁻³m NaCl, 1  × 10⁻³m EDTA, 1% NP‐40) containing protease inhibitors cocktail and phosphatase inhibitors (Bimake, B15002/B14002). The collected proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then the proteins were transferred to polyvinylidene difluoride membranes (Millipore, #IPVH00010). The membranes were blocked with 5% slim milk (Sangon Biotech, #A600669‐0250) for 1 h at room temperature followed by incubation with indicated primary antibodies. Subsequently membranes were washed in Tris‐buffered saline with Tween‐20 (Sangon Biotech, #A600560‐0500) and incubated for 1 hour at room temperature with indicated peroxidase‐conjugated secondary antibodies (Thermo Scientific, #31430). After that, membranes were wash with several times and the chemiluminescent images of immunodetected bands on the membranes were obtained on the X‐ray films (Fujifilm, SUPER RX‐N‐C) using the enhanced chemiluminescence (ECL) system (Bio Rad, #170‐5061. The primary antibodies and dilution ratio were listed in Table S3 in the Supporting Information.

After indicated treatment, cells were lysed with cell lysis buffer (50  × 10⁻³m Tris–HCl PH 7.5, 150  × 10⁻³m NaCl, 1  × 10⁻³m EDTA, 1% NP‐40) containing protease inhibitors cocktail and phosphatase inhibitors (Bimake, B15002/B14002). For each lysate, supernatants were collected after centrifugation and incubated with appropriate antibodies overnight at 4 °C, followed by protein A/G beads incubation for 4 h (Santa Cruz Biotechnology, CA, USA, #sc‐2002). For IP of Flag‐tagged proteins, anti‐Flag M2 affinity gel (Sigma‐Aldrich, #A2220) was used. After incubation, beads were washed with cell lysis buffer for three times. After that, add propriate volume of 2× loading buffer to the beads and boiled for 15 minutes in 95 °C to get the eluted proteins. Next, immunoblot assays were performed with specific antibodies. The antibodies used for Co‐IP or immunoblot assay were listed in Table S3 in the Supporting Information.