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. 2019 Oct 22;9(26):8392–8408. doi: 10.7150/thno.36838

Figure 3.

Figure 3

E3 ubiquitin ligase RNF41 co-immunoprecipitates with CACYBP. (A) Silver-stained SDS-PAGE gel of protein marker only (lane 1) and the immunoprecipitated complex after a two-step TAP from SFB-CACYBP expressing 293T cells (lane 2). Bands were excised from the gel, trypsin-digested, and analyzed by MALDI-TOF. Identified proteins are indicated by lines. (B) Protein coverage of RNF41 in the MS result. The blue lines under the amino acid sequence indicate the supporting peptides matching RNF41. (C) MS sequencing of the best unique peptide (indicated by a red asterisk in (B)) of RNF41 protein. (D-E) Immunoprecipitation and western blotting experiments were performed using anti-HA agarose on lysates derived from 293T cells exogenously expressing Flag-tagged RNF41 and HA-tagged CACYBP (D), or Flag-tagged CACYBP and HA-tagged RNF41 (E). The asterisk indicates a nonspecific band. (F) An endogenous immunoprecipitation assay was performed using Huh7 cell extract and anti-CACYBP antibody. Rabbit normal anti-IgG was used as a control. Western blotting was performed with the indicated anti-CACYBP or anti-RNF41 antibody. (G-H) Immunoprecipitation and western blotting experiments were performed using anti-HA agarose on lysates derived from 293T cells exogenously expressing Flag-tagged CACYBP and the indicated HA-tagged RNF41 truncated mutants (G), or Flag-tagged RNF41 and the indicated HA-tagged CACYBP truncated mutants (H).