Protein standard

HEK 293-T cells (4 x 10⁶) transiently transfected with the plasmids of interest were used to obtain the nuclear fractions as previously described [6 (link)]. Samples (100 μl) of the nuclear fractions were injected on a Superose 12 10/300 GL column (GE Healthcare), and elution performed as previously described [23 (link)]. Dot-Blot analysis of the fractions (100 μl) was performed using the same conditions than for western blot. The protein standards (GE Healthcare and Sigma) used were: Blue dextran (2000 kDa); ferritin (440 kDa); β-amylase (200 kDa); aldolase (150 kDa); alcohol dehydrogenase (150 kDa); conalbumin (75 kDa); ovalbumin (43 kDa); carbonic anhydrase (29 kDa); lysozyme (14.4 kDa); and ATP (551 Da).

HeLa cell nuclear extracts or FLAG-CEP131-containing protein complexes were applied to a Superose 6 size exclusion column (GE Healthcare) that had been equilibrated with dithiothreitol-containing buffer and calibrated with protein standards (Amersham Biosciences). The column was eluted at a flow rate of 0.5 ml min⁻¹ and fractions were collected.

SEC was performed on the HiPrep 16/60 Sephacryl S-400 HR column (GE Healthcare) equilibrated with SEC buffer (the same extraction buffer as described in [45 (link)], but without NP-40 detergent). A mixture of protein standards ranging from 669 to 44 kDa (GE Healthcare), resuspended in the SEC buffer, were run on the column to produce a calibration curve of molecular weights versus elution volumes. The slope equation of the calibration curve was then used to calculate the molecular weight of the peak ARP6 SEC fractions. Total protein extracts were isolated from 1 gram of the WT and mbd9-1 seedling tissues (without roots) using the same extraction buffer that was used for the ARP6-TAP-tag protein complex purification [45 (link)]. For each run, between 1.8 and 2.0 ml of the protein extract was loaded onto the column and 1 ml fractions were collected. For each sample, two biological replicates of the SEC experiments were performed and gave nearly identical results.

SDS-PAGE was carried out according to the procedure of Laemmli with some modifications [22 (link)]. Briefly, the protein samples were prepared by mixing with 1x final concentration of loading sample buffer (0.1 M tris-HCl, pH 6.8, 6% sodium dodecyl sulphate, 30% Glycerol, 15% 2-mercaptoethanol and 0.01% bromophenol blue). Prior to loading to the gel, the samples were heated in a boiling water bath for 5 min. The discontinuous gel system usually had 5% stacking and 12.5% resolving gel. Electrophoresis was carried out using Laemmli buffer at constant current of 15 mA first, till the samples entered into the resolving gel and then at 20 mA till the completion. On completion of electrophoresis, gel was immersed in 0.05% Coomassie Blue R250 in methanol: acetic acid: water (4:1:5) with gentle shaking and was then destained in destaining solution (staining solution without dye) till the background was clear. The protein standards (GE Healthcare, UK) used were phosphorylase b, rabbit muscle (97,000 Da), albumin, bovine serum (66,000 Da), ovalbumin, chicken egg white (45,000 Da), carbonic anhydrase, bovine erythrocyte (30,000 Da), trypsin inhibitor, soybean (20,100 Da), and α lactalbumin, bovine milk (14,400 Da).

rHDL was prepared from purified rh-apoA-I using the Jonas sodium cholate dialysis method with a molar ratio of apoA-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine:cholesterol of 1:100:10 (79 (link)). rHDL particles were purified by gel filtration chromatography with the use of a Sephacryl S300 column (GE Healthcare) on a Bio-Rad Biologics DuoFlo FPLC (Bio-Rad), as previously described (11 (link)). The size of rHDL containing apoA-I was estimated from 4 to 20% nondenaturing equilibrium gel electrophoresis (using precast gels; Bio-Rad) by comparison with protein standards of known Stokes diameter (GE Healthcare) as previously described (77 (link)). Nondenaturing gels were then analyzed by Coomassie blue staining. The amount of cholesterol was determined using a LiquiColor enzymatic cholesterol test kit (Stanbio Laboratory). Total phospholipid content in the rHDL was quantified by microphosphorous assay (80 (link)).

Oligomerisation of full-length VanS_SC solubilised in DPC detergent micelles was analysed using analytical gel filtration chromatography on a 10/300 mm Superose 12 column (GE Healthcare) attached to an AKTA Prime system (GE Healthcare). Equilibration of the column was performed with Buffer A (Buffer A: 20 mM HEPES, pH 7.8, 200 mM NaCl, and 0.075% DPC) before protein samples were loaded. Separation was accomplished at a flow rate of 0.3 mL/min under isocratic conditions until a volume corresponding to 1.5 column bed volumes (36 mL) was collected in 0.3 ml fractions. Protein absorbance was recorded at 280 nm and 254 nm wavelengths, and the elution volume (Ve) of VanS_SC was determined and compared to those obtained for a set of protein standards (GE Healthcare) under identical conditions to yield an estimation of molecular weight. The standards used were as follows: were aprotinin (‘Apro’, 6.5 kDa); ribonuclease A (‘Ribo’, 13.7 kDa); carbonic anhydrase (‘CA’, 29 kDa); bovine serum albumin (‘BSA’, 66.9 kDa); and aldolase (‘Aldo’, 158 kDa).

A375 nuclear extracts and FLAG-USP7-containing protein complexes were applied to a Superpose 6 size exclusion column (GE Healthcare) that had been equilibrated with dithiothreitol-containing buffer and calibrated with protein standards (Amersham Biosciences). The column was eluted at a flow rate of 0.5 ml/min and fractions were collected.