Biochemistry and Immunological methods

Prior to immunofluorescence, 7.000 cells were seeded in the well of a 96-well plate. One day later, cells were transfected with plasmids. 48 hours post-transfection, cells were fixed with 4 % formaldehyde in PBS++ for 20 min. After fixation, cells were washed twice with PBS++

and permeabilized with 0.5 % Triton X-100 in PBS ++ for 10 min. Cells were washed twice for 5 min with PBS++ and incubated in blocking solution (10% FCS) for 10 min. First antibody diluted in blocking solution was added to the well and incubated for 1 hour. After washing three times with PBS++ (2 x shortly and once for 5 min), secondary antibody and Hoechst (0.2 μl/ml, final conc.) diluted in blocking solution were added to the well and incubated for 45 min.

After incubation, cells were washed three times with PBS++ (2 x shortly and once for 5 min).

All the incubation steps were done at room temperature.

The fluorescent pictures were taken by the BD Pathway system.

4.3.2 Immunoprecipitation

Adherent cells were scraped, and centrifuged at 4 °C (10 min at 3000 rpm). Cell pellet was resuspended in 1 ml pre-cooled IP lysis buffer, and homogenized by pushing it 5 times through a 26 G insulin syringe. Cell lysate was centrifuged at 4 °C (15 min at 14.000 rpm) to get rid of cell debris. Supernatant was transferred into a new tube, mixed with 50 μl pre-equilibrated Protein-G-Sepharose (PGS) and incubated at 4 °C for 1 hour on a rotator. Protein/sepharose mixture was centrifuged at 4 °C (4 min at 14.000 rpm), and the supernatant was transferred to a new tube. 30 μl of the lysate was taken as an input control, mixed with 30 μl 6 x Laemmli, boiled for 5 min at 95 °C, and stored at -20°C. The rest was divided into two parts, mixed with 1 μg of each antibody (one specifc antibody and one control antibody) and incubated at 4 °C for 2 hours in a rotator. After centrifugation at 4 °C (15 min at 14.000 rpm), supernatant was transferred to a new tube, mixed with 15 μl PGS and 35 μl sepharose, incubated at 4 °C for 1 hour in a rotator and then centrifuged at 4 °C (2 min at 3.000 rpm). PGS pellet was washed with 800 μl IP lysis buffer by inverting 15 times at 4 °C. After centrifugation (2 min at 3.000 rpm), PGS pellet was washed five times with 800 μl IP buffer, and twice with 500 μl IP buffer

by inverting 15 times at 4 °C. The PGS pellet was finally mixed with 50 μl 6 x Laemmli buffer, heated at 95 °C for 5 min, and stored at -20 °C.

4.3.3 Western blotting analysis

Adherent cells were either scraped in the culture medium, centrifuged and then lysed in the lysis buffer, or directly lysed in the culture plates by applying the lysis buffer after removing medium and washing. Cell lysates were vigorously shaken for 15 min at 4 °C, and centrifuged for 15 min at 4 °C. Supernatant was transferred to a new tube. Protein concentrations were measured by BCA protein assay. Samples were stored at -20 °C. Before loading samples to SDS-PAGE gels, samples were mixed with 6 x Laemmli buffer and incubated for 5 min at 95 °C.

SDS-PAGE electrophoresis

SDS-PAGE is a method of separating proteins in a polyacrylamide gel according to their electrophoretic mobility.

Gels were prepared as follows:

Resolving gel (10 ml) 10% 12% 15%

H2O 4 ml 3.3 ml 2.3 ml

Acrylamide (30%) 3.3 ml 4 ml 5 ml

1.5 M Tris (pH 8.8) 2.5 ml 2.5 ml 2.5 ml

10% SDS 100 μl 100 μl 100 μl

10% APS 100 μl 100 μl 100 μl

TEMED 4 μl 4 μl 4 μl

After pouring resolving gel, 3 ml 100% isopropanol was used to obtain a horizontal surface.

Stacking gel (5 ml) 10%

H2O 3.435 ml

Acrylamide (30%) 830 μl 1.5 M Tris (pH 8.8) 630 μl

10% SDS 50 μl

10% APS 50 μl

TEMED 5 μl

After polymerization, samples were loaded into the wells. Electrophoresis was performed at 80 V in 1 x SDS-PAGE running buffer. When the protein samples passed the border between the two parts of the gel, higher voltage (ca.140 V) was applied.

Western blot

After desired separation was obtained, proteins were transferred onto a nitrocellulose membrane by western blot. Wet blot was performed in transfer buffer for 1.5 h at 100 V in a Biorad Chamber. The quality of transferring was checked by staining membranes with Ponceau S solution.

Immunostaining

After blotting, the membrane was blocked in 5% milk dissolved in PBS-T or TBS-T (called

“milk”). The membrane was then incubated with primary antibody diluted in milk for 2 hours or overnight (4 °C). Afterwards, the membrane was washed with PBS-T or TBS-T for three times for 7 min, followed by incubation with secondary HRP-conjugated antibody for one hour.

The membrane was washed again with PBS-T or TBS-T for three times for 7 min. All washing and incubation steps were performed with gentle shaking. Finally, 1:1 mixture of Luminol/Enhancer solution and peroxide solution of SuperSignal West Dura or Femto (Pierce) was poured to the membrane, and the emitted light signal was detected by X-ray film.

4.3.4 Dual-luciferase reporter assay

In our study, this assay was used to analyze microRNA function on predicted microRNA binding sites. The firefly luciferase reporter, which was used to evaluate microRNA function, contains 3’UTR sequences of predicted target genes. The Renilla (Renilla reniformis, also known as sea pansy) luciferase reporter was used for transfection normalization. In this assay, the activities of firefly and Renilla luciferases are measured sequentially from a single sample.

The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is initiated simultaneously by adding Stop &

Glo® Reagent to the same sample.

4.3.5 BCA protein assay

This assay is based on the biuret reaction, in which Cu²⁺was reduced to Cu⁺by protein in an alkaline medium. Bicinchoninic acid (BCA) then reacts with the reduced cation. The BCA/copper complex is water soluble and exhibits a strong absorbance at 562 nm.

We use BCA protein assay kit from Pierce. First, a fresh set of BSA standards (diluted in the same solution as the samples) was prepared. Then, the BCA working reagent was prepared by mixing 50 parts of BCA Reagent A with 1 part of Reagent B. 5 μl of the samples and BSA standards were mixed with 40 μl working reagent, and incubated for 30 min at 37 °C. After incubation, samples and BSA standards were measured by using Nanodrop (Peqlab) BSA method.