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Proteins extracts were prepared from equal number of Tos4-GFP cells in asynchronous culture grown in supplemented EMM media, after treatment with 12 mM hydroxyurea (HU), and after washing twice with media for release from HU. Cells in mid-log phase were harvested and whole-cell protein extract was prepared by vortexing acid-washed glass beads in 20% trichloroacetic acid (TCA) and washing beads with 5% TCA. Lysates were boiled for 5 min in Laemmli Sample buffer (4%SDS, 60 mM Tris-HCl, pH 6.8, 5% glycerol, 4% 2-mercaptoethanol, 0.01% bromophenol blue) and analyzed by 4–12% SDS-PAGE (Expedeon), followed by immunoblotting with rabbit anti-GFP (Abcam 290; 1:1000) and rabbit anti-cdc2 (gift from Nurse lab; 1:4000) as loading control. After secondary antibody (anti-rabbit Alexa Flour 488; 1:4000) incubation, blots were developed using Amersham Typhoon biomolecular imager.

CPS and TetH_c conjugation was performed as detailed previously [16] (link), [17] (link). Briefly, purified CPS was solubilised at 5 mg/mL in PBS (Sigma) and sodium meta-periodate (Alpha Aesar) was added to a final 30 mM concentration. The mixture was gently stirred at room temperature for 40 min and afterwards, excess oxidising agent was removed by dialysis (GeBAflex-tube maxi). To the dialysed polysaccharide solution, TetH_c protein was added to a final concentration of 5 mg/mL in PBS (Sigma). Sodium cyanoborohydride (Sigma: 1 M NaBH₃CN in 10 mM NaOH) was then added at 10 μL per mL of conjugation mixture and then stirred at room temperature for 4 days. Following this, a further 10 μL of sodium borohydride (Sigma: 1 M NaBH₄ in 10 mM NaOH) was added to each mL of conjugation mixture and stirring was continued at room temperature for 40 min. The conjugate reactions were then dialysed against distilled water (GeBAflex-tube maxi) and analysed by SDS-PAGE (12%, Expedeon) and western blot using anti-CPS monoclonal antibody DSTL189. The resulting products were then re-dissolved in ultrapure water and stored at −20 °C.

Sf21VM Cells were maintained in ExCell420 Medium in Erlenmeyer culture flasks shaking at 27.5°C. Human COP9 signalosome subunits bearing N‐terminal Strep(II) or His6 tags were co‐expressed by co‐infection of Sf21VM cells with three baculoviral vectors obtained from Lingaraju et al (2014). After 48 h, cells were mildly lysed and COP9 signalosome subunits and complexes differentially affinity‐purified on StrepTactin and Ni‐NTA‐coated magnetic beads (Qiagen) followed by bead boiling in SDS loading buffer and subunit detection via SDS–PAGE and InstantBlue staining (Expedeon). Subunits were identified by size and in reference to individual expression and in‐gel detection.