Figure 8.

TRIM21–Tn3 fusion proteins degrade untagged and endogenous POI.A and B, immunoblot analysis of cell lysate from HEK 293F cells cotransfected with a plasmid encoding MALT1 (A) or EED (B) and a plasmid encoding the indicated TRIM21–Tn3 variant. C, immunoblot analysis of cell lysate from HEK 293F cells transfected with a plasmid encoding the indicated TRIM21–Tn3 variant or an empty plasmid (“empty”). Cells were harvested and lysed 72 h after transfection. Blots were probed with an anti-MALT1 antibody (A and C) or an anti-EED antibody (B), and an anti-FLAG antibody was used to detect TRIM21–Tn3. An equivalent amount of total protein was added to each lane, and blots were probed with an anti-β-actin antibody to confirm equivalent loading. Immunoblots are representative of two replicate experiments. Immunoblot images for biological replicates are shown in Fig. S9. D–F, relative quantitation of total MALT1 (D and F) or EED (D) levels across biological replicates by densitometry using ImageJ software. MALT1 or EED band intensity was normalized to the band intensity of β-actin and then divided by the normalized band intensity of the control sample for each replicate. Statistical significance was determined by two-tailed paired Student’s t test. Statistical data are shown in Table S4. ∗p ≤ 0.05 and ∗∗p ≤ 0.01. EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; POI, protein of interest; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21.