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. 2024 Jan 11;134(5):e166149. doi: 10.1172/JCI166149

Figure 6. TRIM56 promotes FASN degradation.

Figure 6

(A) HepG2 cells were transfected with empty vector or FLAG-TRIM56 in the presence of indicated compounds. FASN protein expression was determined (n = 3). (B) HEK293T cells were transfected with empty vector or FLAG-TRIM56 in the presence of MG132 before IP was performed (n = 3). (C) HEK293T cells were transfected with HA-FASN with or without FLAG-TRIM56 and MYC-ubiquitin before IP was performed (n = 3). (D) HEK293T cells were transfected with HA-FASN in the presence or absence of FLAG-TRIM56 and MYC tagged site-specific ubiquitin mutants. Then, IP was performed (n = 3). (E) HEK293T cells were transfected with HA-FASN with or without FLAG-TRIM56 and MYC-tagged active K48–linked ubiquitin (K48O) or inactive K48–linked ubiquitin (K48R) mutant. Then, IP was performed (n = 3). (F) HEK293T cells were transfected with HA-FASN in the presence or absence of FLAG-TRIM56 (full-length), FLAG-TRIM56 (1–521 aa), and MYC-ubiquitin (UB). Then, IP was performed (n = 3). (G) HEK293T cells were transfected with HA-FASN with or without FLAG-TRIM56, E3 ligase–defective mutant FLAG-TRIM56 (21AACC24), and MYC-UB. Then, IP was performed (n = 3). (H) Intracellular TG content (n = 6). ***P < 0.001, by 1-way ANOVA followed by Bonferroni’s post hoc test. (I) HepG2 cells were transfected with empty vector (FLAG) or FLAG-TRIM56, or FLAG-TRIM56 mutant (21AACC24) with or without PO before Nile Red staining (n = 3). Scale bar: 50 μm. (J) HepG2 cells were transfected with empty vector (FLAG) or FLAG-TRIM56 mutant (21AACC24) to determine the expression of the indicated proteins (n = 3). (K) Expression of the indicated genes in HepG2 transfected with empty vector (FLAG) or FLAG-TRIM56 mutant (21AACC24) in the presence of PO (n = 5). Two-tailed Student’s t test for SCD1 and GPAM; Mann-Whitney U test for ELOVL1, ELOVL6, and DGAT2.