Methods of protein biochemistry

Protein extraction was performed in UREA buffer. Therefore, cells were places on ice and 300 µL buffer were added to each well of a 6 well plate. Cells were detached with a cell scraper and the cell suspension was collected in a protein low binding tube. Next, the cell suspension was

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sonicated in a Bioruptor for four high power sonication cycles (15 sec ON, 45 sec OFF) at 4 °C.

Afterwards, cell debris was spun down at 14,000 g for 3 min at 4 °C. The supernatant was recovered and an aliquot was taken to determine protein concentration by Bradford assay.

2.2.4.2 Bradford protein assay

Protein extracts obtained as described above, were first diluted 1:4 in H2O. Then, protein concentration was measured with Pierce Coomassie (Bradford) protein assay kit. Therefore, 20 µL of albumin protein standards with defined concentrations (100 ng/µL, 250 ng/µL, 500 ng/µL, 750 ng/µL, 1 µg/µL) were mixed with 1 mL of 1x Bradford reagent, incubated at RT for 5 min and analyzed in a spectrophotometer to obtain a standard curve. Afterwards, 20 µL of protein sample were mixed with 1 mL of 1x Bradford reagent and protein concentration was determined with a spectrophotometer by use of the previously obtained standard curve.

2.2.4.3 SDS-PAGE

Samples containing 3-20 µg of protein in 1x SDS loading buffer were boiled at 95 °C for 5 min before they were loaded on self-casted 15 % SDS gels which were prepared according to this recipe:

Reagent 15% SDS

separating gel

5% SDS stacking gel

H2O 1.4 mL 2.2 mL

30 % Acrylamide/Bis (37.5:1) 3 mL 0.5 mL

Tris (1.5 M, pH 8.8) 1.5 mL 0.25 mL

20 % SDS 0.03 mL 0.015 mL

10 % APS 0.06 mL 0.03 mL

TEMED 0.005 mL 0.003 mL

Final volume 6 mL 3 mL

Protein samples were run at 90 V in the stacking and the separating part of the gel until the bromophenol blue dye band reached the bottom of the SDS gel.

2.2.4.4 Western blot

Right after SDS-PAGE the stacking part of the SDS gel was removed and semidry protein transfer to a PVDF membrane was performed in Towbin buffer with a western blotting transfer system at 25 V, 1 A for 30 min. Afterwards, the efficiency of the transfer was determined by

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Ponceau staining of the membrane for 5 min at RT and/or by coomassie staining of the gel for 5 min at RT.

In case a Ponceau staining was performed, the staining was rinsed off the membrane with TBS before it was blocked in TBS, 3 % milk, 0.05 % Tween 20 for one hour at RT, 35 rpm. After blocking, the membrane was cut in two parts at the band of 35 kDa of the PageRluer Plus Prestained ladder. The lower part of the membrane (10-35 kDa) was treated with anti-V5 antibody (1:5000) in blocking solution and the upper part of the membrane (35-250 kDa) was treated with anti-α-Tubulin antibody (1:4000) in blocking solution O/N at 4 °C, 35 rpm.

Next day, membranes were washed three times 10 min in TBS, 0.05 % Tween 20 at RT, 35 rpm. Afterwards, TBS, 0.05 % Tween 20 was removed and the secondary antibody was added in TBS, 3 % milk, 0.05 % Tween 20 for two hours at RT, 35 rpm. Anti-V5 stained lower part of the membrane was treated with anti-rabbit HRP (1:5000) and anti-α-Tubulin stained upper part of the membrane was treated with anti-mouse HRP (1: 10,000). Then, membranes were washed three times 10 min in TBS, 0.05 % Tween 20 at RT, 35 rpm. In the meantime, homemade ELC reagent was prepared like this:

ELC reagent Amount

Tris (1.5 M, pH 8.5) 10 mL

H2O2 2.6 µL

Coumaric acid 25 µL

Luminol 50 µL

Final volume ~10 mL

The membrane was covered with ELC reagent for 1 min and chemiluminescence was analyzed with a chemiluminescence imager.

2.2.4.5 Mass spectrometry

First, 100 µg of proteins were reduced in DTT (2 mM) for 30 min at 25 °C and subsequently free cysteines were alkylated in iodoacetamide (11 mM) for 20 min. Digestion with Lys-C (1:40 w/w) was carried out at 30 °C for 18 h under gentle shaking. Afterwards, the sample was diluted three times with ammonium bicarbonate solution (50 mM) and 7 µL of immobilized trypsin were added before the sample was incubated at 30 °C for 4 h under rotation. Then, 10 µL trifluoroacetic acid were added and trypsin beads were removed by centrifugation. 15 µg of digested protein were desalted on STAGE Tips, dried and dissolved in 20 µL acetic acid in water (0.5 %) [847].

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For the analysis, 5 µL of digested sample were injected in duplicates on a LC-MS/MS system using a 240 min gradient from 5 % to 45 % solvent B (80 % acetonitrile, 0.1 % formic acid) into solvent A (5 % acetonitrile, 0.1 % formic acid). A 30 cm capillary with 75 µm inner diameter was packed with 1.8 µm C18 beads for chromatographic separation. At one end, a nanospray tip was generated with a laser puller, allowing fretless packing.

The nanospray sources was operated with a spray voltage of 2.1 kV and an ion transfer tube temperature of 260 °C. A data dependent mode with a top10 method was chosen for data acquisition on the Q Exactive Plus: a survey MS scan with a resolution of 70,000 at m/z 200 was followed by up to ten MS/MS scans on the most intense ions with an intensity threshold of 5,000. When selected once for fragmentation, ions were excluded from selection for 30 sec to increase new sequencing events.

The MaxQuant proteomics pipeline with the Andromeda search engine and Uniprot database built in was used for data analysis [848]. Carbamidomethylation of cysteines was selected as fixed modification, while oxidation of methionine and acetylation of the N-terminus were selected as variable modifications. Parameters were left as default except for the search engine peptide assignments, which were filtered at 1 % FDR and the feature match between runs, which was enabled.

2.2.4.6 Luciferase reporter gene assay

Homemade luciferase reporter assay reagents were used to detect luciferase activities with Centro LB 960 Microplate luminometer (Berthold) and MikroWin software package (Mikrotek Laborsysteme). Per sample, 10 µL of lysate or 9 µL of in vitro translation reaction mix were treated with 45 µL of FLuc reagent (75 mM Hepes pH 8.0, 0.1 mM EDTA, 4 mM MgSO4, 530 µM ATP, 270 µM Coenzyme A, 470 µM DTT, 470 µM luciferin) and 45 µl of RLuc reagent (2.2 mM Na2EDTA, 220 mM K3PO4 pH 5.1, 0.44 mg/mL BSA, 1.1 M NaCl, 1.3 mM NaN3, 0.6 µg/mL colenterazine).

3R^ESULTS

3.1. Generation and characterization of mouse embryonic stem cells expressing inducible