Figure 5.

Immunoblot analysis of MALT1 and EED degradation.A and B, immunoblot analysis of cell lysate from HEK 293F cells cotransfected with a plasmid encoding MALT1-tGFP (A) or EED-tGFP (B) as well as a plasmid encoding the indicated TRIM21–Tn3 variant. Cells were harvested and lysed 72 h after transfection. Blots were probed with an anti-MALT1 antibody (A) or an anti-EED antibody (B) as well as an anti-FLAG antibody to detect TRIM21–Tn3. An equivalent amount of total protein was added to each lane, and blots were also 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. S5, A and B. Bands for MALT1-tGFP and MALT1 are present in A because HEK 293F cells natively express MALT1 (93, 94). C and D, relative quantitation of total MALT1 (C) or EED (D) levels across two biological replicates (Fig. S5A) by densitometry using ImageJ software. 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. Data represent the same sample sets analyzed by flow cytometry in Figure 4, B and C. EED, embryonic ectoderm development protein; HEK, human embryonic kidney cell line; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; tGFP, TurboGFP; Tn3, third fibronectin type III domain of human tenascin-C; TRIM21, tripartite motif containing-21.