Protein biochemistry

2.11.1. Whole cell lysates and cell fractionation Whole cell lysates

Cells were harvested by centrifugation at 178 x g for 5 min at 4°C, washed once with PBS and centrifuged for 4 min at 300 x g at 4°C. For whole cell lysates, cell pellets of 1 x 10⁶ to 2 x 10⁶cells were resuspended in 50μL RIPA lysis buffer (supplemented with Protease and Phosphatase Inhibitor) respectively and incubated on ice for 25 min. Subsequently, residual cell debris was lost by centrifuging cell suspensions at 20,000 x g for 15 min at 4°C. Super-natants were transferred to new reaction tubes and total protein amount was measured using Bradford Assay.

Cell fractionation

Cell fractionation has been performed as described by (Schreiberet al., 1989). In short, cell pellets of 5 x 10⁶ cells were resuspended in 400 μl Nuclear Extract Buffer A and incubated on ice for 13 min. Afterwards 25 μL 10 % NP-40 solution was added to each tube, mixed thoroughly for 5 seconds and immediately centrifuged at 15.800 x g for 5 min at 4°C. The supernatant containing the cytosolic fraction was transferred to a new tube. The pellets were resuspended in 50μl Nuclear Extract Buffer B and shaken thoroughly for 1-4 h at 4°C. After centrifugation at 15.800 x g for 5 min at 4°C the supernatant, containing the nuclear fraction, was transferred to a new tube and protein amount of both fractions was measured using Brad-ford assay.

2.11. PROTEIN BIOCHEMISTRY 43

2.11.2. Determination of protein concentration by Bradford assay

Determination of protein concentration was carried out by photometrical analysis using a mod-ified Bradford assay (Bradford, 1976). A BSA standard curve was prepared and protein sam-ples were diluted 1:200. 100 μL of Bradford solution was added to 50 μL diluted samsam-ples and BSA standards and incubated for 10 min at RT. Measurement was performed at 620 nm with Thermo Multiscan Ex Platereader. Protein concentration was calculated, adjusted with RIPA to 1 μg/μL and boiled for 5 min at 95°C in 6x Laemmli Buffer. Samples were stored at -20°C.

2.11.3. SDS PAGE and Western Blotting SDS PAGE

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was used for sep-aration of proteins according to molecular weight (Laemmli, 1970). Modified buffers listed in table 2.3 were used to prepare a gel composed of a 5% stacking and a 10-15% separation gel.

15 -25 μg total protein per sample was loaded onto the gel and electrophoresis was performed in 1x Running buffer at constant 15 mA per gel for the stacking gel and 30 mA per gel for the separation gel.

Western blotting and immunodetection

Separated proteins were transferred from SDS gel onto a hydrophobic PVDF membrane using wet tank sandwich method (Renart et al., 1979; Towbin et al., 1979). The membrane was activated for 30 sec in 100% MeOH, rehydrated in H₂O for 2 min and equilibrated in transfer buffer prior to use. Membrane and gel were stacked between filter papers into a wet tank chamber filled with ice cold transfer buffer. Protein transfer was performed at 4°C and 100V for 1 h. Effective transfer of proteins was visualized by ponceau S staining and unspecific binding sites were blocked using 5% BSA-TBS-T for 1 h at RT. After blocking, antibody solutions listed in table 2.8 were added over night at 4°C. Next day, blots were washed four times 5 min with 1x TBS-T. The secondary HRP coupled antibody against the species of origin of the first antibody was added in a dilution of 1:10,000 in 5% BSA-TBS-T for 1 h at RT. The membrane was washed four times for 5 min with TBS-T, before detection of bound antibody using Western lightning Plus ECL Solution and Western Lightning Image Reader. For reblotting, the membrane was stripped of bound antibody for 20 min with 1x ReBlot mild buffer, blocked in 5% BSA-TBS-T for 60 min at RT and another primary antibody was added over night as described before.

44 2| Materials and Methods 2.11.4. Ago2-RNA immunoprecipitation

The protocol for Ago2-RNA Immunoprecipitation (Ago2-RIP) used in this thesis was based on the paper “Systematic Analysis of Viral and Cellular MicroRNA Targets in Cells Latently In-fected with Humanγ-Herpesvirus by RISC Immunoprecipitation Assay” (Dölkenet al., 2010).

1.5 x 10⁸cells per replicate were harvested and washed twice in cold PBS before lysis in 4.2 mL Ago2-RIP lysis buffer in DNAlo bind reaction tubes. DTT, protease inhibitors and RNase-Out were prepared freshly and added immediately before use. Lysates were incubated for 20 min on ice, frozen at -80°C for 10 min and thawed for 8 min at 30°C and 300 rpm. Cell lysates were cleared by centrifugation at 15,000 x g for 30 min at 4°C. An aliquot was taken as input control for Western blotting. Supernatants were supplemented with 4.5 mg mag-netic beads that were coupled to 3.6 μg rat anti human Ago2 antibody or isotype control over night at 4°C and washed once with Ago2-RIP wash buffer supplemented with protease and RNase inhibitors. After incubation for 2.5 h rotating at 4°C, an aliquot from the supernatant was taken as depletion control for Western blotting and the beads were washed 5 times with ice cold Ago2-RIP wash buffer supplemented with protease and RNase inhibitors followed by two wash steps with ice cold PBS supplemented with RNase inhibitor. An aliquot of beads was taken for Western blotting output control. RNA was recovered from the remaining beads by adding 1 mL Qiazol. Total RNA was prepared using the miRNeasy kit (QIAGEN) following the manufacturer’s instructions including DNase digestion.

CDNA library preparation and Next Generation Sequencing were performed at the GMAK, HZI Brunswick using Illumina kits and technology. For mRNA sequencing, poly A enrichment was applied (ScriptSeq™ v2 RNA-Seq Library Preparation kit, Illumina). For small RNA sequencing no enrichment was performed (TruSeq Small RNA Library Preparation kit). For sequencing library pool of 12 pM was multiplexed on a single lane. Cluster generation was performed with cBot (Illumina) using TruSeq SR Cluster Kit v3–cBot-HS (Illumina). Samples were sequenced (50 bp single-end) on the Illumina High Seq 2500 using TruSeq SBS Kit v3 - HS (Illumina) for 50 cycles.

RNA sequencing reads were pre-processed including trimming of reads (fastq-mcf, ea-utils) and quality control (FastQC) ensuring high quality reads and removal of adapter sequences.

Trimmed reads were mapped to the human genome (hg38) by STAR (Dobinet al., 2013) and counted by HTseq (Anderset al., 2015). Further analysis was performed using the software environment R/Bioconductor and the respective software packages. Normalization was per-formed according to total number of reads within a sample and between samples (Anderset al., 2010). Thresholds for differentially expressed genes were set to at least 2-fold enrichment and a Benjamini-Hochberg adjusted p-value lower than 0.05.