Research Paper Volume 12, Issue 3 pp 2507—2529

Figure 5. TRIM21 interacts with PRMT5 in U2 OS cells. (A) Colocalization of TRIM21 and PRMT5 was observed in U2 OS cells using antibodies against TRIM21 (green) and PRMT5 (red). Scale bar = 20 μm. (B) DAPI was used to indicate nuclei. Myc-VN155-PRMT5 and HA-VC155-TRIM21, along with HA-cerulean, were cotransfected into U2 OS cells for 48 h, and the reconstituted Venus fluorophore (yellow, arrows) was visualized via confocal microscopy. Scale bar = 20 μm. (C) The endogenous interaction between TRIM21 and PRMT5 was validated using a co-IP assay. (D, E) shRNAs targeting TRIM21 (shT#1 and shT#2) or plasmid encoding HA-TRIM21 were applied to knock down or overexpress TRIM21, and the protein and mRNA levels of TRIM21 and TXNIP were then determined by WB or quantitative real-time PCR, respectively. (F) Dox-inducible TRIM21-expressing cells was treated with MG132 (10 μM) for 12h, the protein expression of TRIM21, TXNIP, and p21 was then determined by WB.