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. 2006 Jun 30;103(28):10735–10740. doi: 10.1073/pnas.0600397103

Fig. 4.

Fig. 4.

HERC5, together with Ube1L and UbcH8, induces the FLAG–ISG15 conjugation in HeLa cells without type I IFN treatment. (A) HeLa cells were transfected with expression vector encoding for FLAG–ISG15 plus vectors encoding Ube1L (lane 2), UbcH8 (lane 3), Ube1L and UbcH8 (lane 4), or Ube1L, UbcH8, and HERC5 (lanes 5). Twenty-four hours after transfection, total lysates were prepared, and FLAG–ISG15 conjugates were detected with anti-FLAG antibody. (B–I) HeLa cells were transfected with expression vector encoding for FLAG–ISG15 (lane 1), FLAG–ISG15 plus vector encoding Ube1L and UbcH8 (lane 2), or FLAG–ISG15 construct together with vectors encoding Ube1L, UbcH8, and HERC5 (lanes 3). Total lysates were harvested, FLAG–ISG15 conjugates were immunoprecipitated with anti-FLAG M2-conjugated agarose, and protein complexes were separated in SDS/PAGE and then immunoblotted with antibodies against destrin (B), cofilin (C), enolase (D), GAPDH (E), Hsp27 (F), Hsc70 (G), peroxiredoxin I (H), and peroxiredoxin VI (I).