Phosphoprotein purification kit

After CycD–CDK phosphorylation of KRP4;2, proteins were heat-denatured, loaded to the PhosphoProtein Purification Kit column (Qiagen) and incubated for 30 min at 4 °C in the purification column. Then proteins were eluted, collected, and this procedure repeated five times. In every purification, the amount of resin used was calculated to purify around 25 µg of recombinant p-His-KRP4 protein; column washing conditions were as indicated by the manufacturer, collecting 500 µl fractions. The final elutions were desalted and then were pooled and concentrated by means of Amicon Ultra and protein was quantified. Identification of the phosphorylated protein was achieved by western blot, using the anti-pT-P/pS-P antibody (1:1000 dilution) (see Supplementary Fig. S8A, B). Quantification of p-KRP4;2 protein was by the method of Bradford (1976) (link).

Cell phosphoprotein isolation was carried out using a phosphoprotein purification kit (Qiagen, Hilden, Germany). Briefly, cells or nuclear fractions were washed with HEPES buffer and scraped off for lysis in CHAPS buffer, after which 2.5 mg of total protein lysate was subjected to purification on the columns, and phosphoproteins were then eluted and quantified.

Asynchronous or mitotic cells were prepared in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 1% (v/v) triton X-100 and Complete EDTA-free protease inhibitor cocktail (Roche)) and lysates were cleared by 15,000 g centrifugation for 15 min at 4 °C. 100 to 200 μg of proteins were then incubated in a 100 μl final volume including either 3 U/μl Calf Intestinal Alkaline Phosphatase (CIP, 37 °C for 3 h, New England BioLabs) with 1× buffer, or 8 U/μl Lambda Phosphatase (λPP, 30 °C for 3 h, New England BioLabs) with 1× buffer and 1 mM MnCl₂. For control conditions, the same quantity of each phosphatases was inactivated according to the manufacturer instructions, and then added with proteins, 50 mM EDTA, 50 mM NaF and 50 mM Na₃VO₄ to the 100 μl final volume sample mix. In vitro assayed extracts were completed with Laemmli sample buffer and heated at 100 °C for 10 min to denature proteins before western blotting analysis.
Phosphorylated proteins were also purified by affinity chromatography. For this purpose, lysates from asynchronous or mitotic extracts were submitted to a phosphoprotein purification kit (Qiagen) following the manufacturer instructions. Total cell extracts, flow through and eluates were analysed by western blotting to control and characterize phosphoprotein enrichment.

Total protein was extracted from cells in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulphate) supplemented with a protease and phosphatase inhibitor cocktail; protein lysate was combined with SDS loading buffer (4% SDS, 0.2% bromophenol blue, 20% glycerol, 5% 2-mercaptoethanol) and boiled for 5 minutes. Protein samples were run on Bis-Tris (Novex) gels and transferred to nitrocellulose. Blots were blocked in 50% Seablock blocking buffer (Thermo Scientific, diluted in PBS) and incubated with primary antibody overnight in blocking buffer. Antibodies used were to HCC1/RBM39 (Atlas, HPA001591), SIN3B (Santa Cruz, sc-768), Phospho-SMAD1/5 (Ser463/465) (Cell Signaling Technologies, 9516) and beta tubulin (Sigma T5283). Secondary antibodies used were IRDye 680RD Donkey anti-Rabbit IgG and IRDye 800CW Donkey anti-Mouse IgG (LI-COR), incubated for 1 hour in blocking buffer. Blots were imaged on a LI-COR Odyssey CLx. Nuclear and cytoplasmic fractions were extracted with an NE-PER kit (Thermo Scientific), to the manufacturer’s instructions. Separation of cellular fractions was confirmed by blotting for anti-Histone H3 (Abcam, 131711). Phosphorylated and unphosphorylated fractions from C2C12 cells were extracted with a PhosphoProtein Purification Kit (Qiagen) to the manufacturer’s instructions.

The Ser/Thr phosphoprotein was purified, according to the manufacturer's instructions by using PhosphoProtein Purification Kit (Qiagen No. 37101). Take a volume of lysate containing 2.5 mg of total protein, and adjust the protein concentration to 0.1 mg/ml. Finally, 30 μl concentrating protein was used for western blot.