Fig 2: Trans-SUMOylation of non-fused TTR occurs via association with Ubc9_TTR or Ubc9mut_TTR.TTR fused to the C-terminus of non-mutated (U_T) (A) or the K14,153,154R mutant of Ubc9 (Umut_T) (B) was co-expressed with EGFP-labelled SUMO-1 (ES1) or SUMO-3 (ES3) in the absence or presence of non-fused HA-labelled TTR (HA_TTR). The cell lysates were analysed by WB using anti-TTR antibodies (aTTR). The asterisks indicate truncated SUMOylated proteins. ES conjugates and X conjugates represent proteins modified by EGFP-labelled SUMO or endogenous modifiers, respectively.

Fig 3: Mutation K126R of TTR alters the SUMOylation patterns of TTR_Ubc9mut.Non-mutated TTR (T) or K126R mutant of TTR (T126), fused to either the K14,153,154R mutant of Ubc9 (Umut) at the N-terminus (T_Umut and T126_Umut) (A) or the C-terminus (Umut_T and Umut_T126) (B) was co-expressed in HEK293 cells with EGFP-labelled SUMO-1 (ES1) or SUMO-3 (ES3). The cell lysates were analysed by WB using anti-TTR antibodies (aTTR). Arrows with letters (a, b, c, d, or e) indicate band differences caused by the K126R mutation of TTR.

Fig 4: K126R mutation of TTR in TTR_Ubc9 but not in Ubc9_TTR alters the SUMOylation pattern.Non-mutated TTR_Ubc9 (T_U) and Ubc9_TTR (U_T) or the K126R mutant of TTR in TTR126_Ubc9 (T126_U) or Ubc9_TTR126 (U_T126) were co-expressed in HEK293 cells with EGFP-labelled SUMO-1 (ES1) or SUMO-3 (ES3). The cell lysates were analysed by WB using anti-TTR antibodies (aTTR). The letters indicate changes in the mobility of the bands resulting from the K126R mutation in TTR. ES conjugates and X conjugates indicate forms of the fusion proteins modified by EGFP-labelled SUMO or endogenous modifiers, respectively.

Fig 5: SUMOylation patterns of TTR_Ubc9 and Ubc9_TTR.TTR fused to the N-terminus (T_U) or C-terminus (U_T) of Ubc9 was co-expressed in HEK293 cells with EGFP-labelled SUMO-1 (ES1), SUMO-3 (ES3) or EGFP (E). Cell lysates were analysed by WB using anti-TTR (A) or anti-Ubc9 antibodies (B). The asterisk indicates truncated SUMOylated proteins. ES conjugates and X conjugates indicate forms of T_U and U_T modified by EGFP-labelled SUMO or endogenous modifiers, respectively.