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. 2024 Mar 1;227(1):iyae033. doi: 10.1093/genetics/iyae033

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Fig. 2. — Interaction between FBF RBD and MPN domain of CSN-5 is conserved in evolution. a) GST or GST::CSN-5 were tested for binding His₆::FBF-1^RBD, His₆::FBF-2^RBD, and His₆::FBF-2^N-term constructs. Input is clarified FBF lysate that was added to GST pulldown. Proteins are detected by Western blot with anti-His and anti-GST; the position of full-length GST::CSN-5 (representative image from His₆::FBF-2^N-term pulldown) is marked with an arrowhead, and a lower band represents GST::CSN-5 degradation product (dot). b) FBF-1/-2 and PUM1/2 truncation constructs. Rectangle: RBD. Amino acid positions of the truncations are indicated above each construct. c) GST::CSN-5 truncation constructs were tested for binding His₆::FBF-2. Input is clarified FBF-2 lysate added to GST pulldown. Proteins are detected by Western blot with anti-His and anti-GST; the positions of GST-tagged constructs are marked with arrowheads, and C-terminally truncated fragments of GST::CSN-5 constructs are marked with dots. d) CSN-5, CSN-6, and CSN5 truncation constructs. Rectangle: core MPN domain. C-terminal helices (ovals) that incorporate into the helical bundle of the COP9 signalosome (Lingaraju et al. 2014). Amino acid positions of the truncations are indicated above each construct. e) GST pulldown of His₆::FBF-2 with GST::CSN-5^MPN and increasing concentrations of GST::CSN-6^MPN. FBF-2 is detected by Western blot with anti-His. Input is clarified FBF-2 lysate added to GST pulldown. Total eluate protein was seen by Coomassie to confirm GST retention on GSH beads; arrowheads mark GST::CSN constructs. Relative concentrations of GST-tagged constructs were determined by total protein staining densitometry of GST fusion bands in the eluates, where GST::CSN-5^MPN was set to 1×, and relative amounts of GST::CSN-6^MPN are indicated above the corresponding lanes. f) GST pulldown of human homologs, GST::CSN5^MPN with His₆::PUM1^RBD or His₆::PUM2^RBD. PUM proteins are detected by Western blot with anti-6xHis. Input is clarified PUM lysate added to GST pulldowns. Total eluate protein (representative image from His₆::PUM2^RBD pulldown) detected by stain-free chemistry to confirm GST fusion retention to GSH beads; arrowhead marks GST::CSN5^MPN.