Journal: Genes & Development
Article Title: Intrinsically disordered regions stimulate concentration of small nucleolar ribonucleoproteins and formation of Cajal bodies and nucleoli
doi: 10.1101/gad.353180.125
Figure Lengend Snippet: Dissection of Nopp140 requirements for interaction. ( A ) Amino acid sequence of human Nopp140. Negatively (red) and positively (yellow) charged amino acids are shaded. The 10 acidic serine stretches are numbered. All serines in those stretches are or become casein kinase 2 consensus sites after phosphorylation. Consensus NLSs are indicated (red lettering). The N and C termini are demarcated from the large central repeat domain (thick black bars). The borders of the repeats tested are indicated by thin black lines. All Nopp140 constructs were expressed fully phosphorylated in baculovirus-transduced Sf9 insect cells. All others were expressed in bacteria. ( B ) MBP-NAP57 (lane 1 ), Nopp140 (lane 2 ), and calf intestinal phosphatase (CIP; bottom band) added to Nopp140 (lane 3 ). Addition of CIP is indicated (+ above the lane). (Lane 2 vs. 3 ) Note the nearly 40 kDa shift in migration upon dephosphorylation of Nopp140. (Lanes 4 , 5 ) MBP-NAP57 was added to Nopp140. Only phosphorylated Nopp140 (lane 6 ) but not (or barely) dephosphorylated Nopp140 (lane 7 ) is pulled down by NAP57. ( C ) MBP-NAP57 and C/D core proteins MBP-NOP56 and MBP-NOP58 pull down the Nopp140 repeat domain alone (lanes 7 – 9 ) but not the control MBP-MCP (lane 10 ). For identification purposes on SDS-PAGE, GFP-Nopp140 fusion constructs were used instead of Nopp140 alone. ( D ) MBP-PAF49 also pulled down full-length Nopp140 (lane 6 ), its repeat domain (R1–10; lane 7 ), or repeats 1–7 (lane 8 ) or 1–3 (lane 9 ). ( E ) Similarly, MBP-NOP58 pulled down repeat domains 1–10 (lane 7 ), 1–7 (lane 8 ), and 1–3 (lane 9 ). ( F ) MBP-NOP58 pulled down the same repeats of Nopp140 (lanes 7 – 9 ) and was additionally tested for repeats 8–10 (lane 10 ) and repeat 1 alone (lane 11 ), all of which were pulled down. ( G ) All tested repeats were only pulled down in a phosphorylation-dependent manner. MBP-PAF49 pulled down repeats 1–10 (lane 7 ), 1–7 (lane 9 ), and 1–3 (lane 11 ) but not after dephosphorylation (lanes 8 , 10 , 12 ). ( H ) Nopp140 alone was not pulled down by MBP-NAP57 after dephosphorylation (lane 6 ), and MBP-MCP did not pull down Nopp140, whether phosphorylated (lane 7 ) or not (lane 8 ). ( I ) Nopp140 before and after CIP treatment (lanes 1 and 2 , respectively) and Nopp140 with 46 CK2 consensus site serines replaced by alanines in the repeat domain (NoppSA, lane 3 ). (Lane 4 ) Surprisingly, CIP treatment of NoppSA further accelerated its mobility, indicating additional phosphorylation sites. Regardless, whereas Nopp140 was efficiently pulled down by MBP-NAP57 (lane 5 ), CIP-treated constructs were not or were only barely pulled down (lanes 6 – 8 ).
Article Snippet: Using the vectors listed in , Nopp140 or fragments thereof were expressed in SF9 cells using the Bac-to-Bac baculovirus expression system (Gibco) following the supplier's protocol.
Techniques: Dissection, Sequencing, Phospho-proteomics, Construct, Bacteria, Migration, De-Phosphorylation Assay, Control, SDS Page
Expression Systems Inc is a verified supplier 
Expression Systems Inc manufactures this product 
