UNIVERSITY OF COIMBRA

Faculty of Sciences and Technology from University of

Coimbra

Masters Program in Cellular and Molecular Biology Cell

Regulation (2016/2017)

Nuclear export activity of the HIV-1 Rev-

protein: dependence on the CRM-1
transporter

Group 9:
Cristiana Ramos
Nuno Beltro
Raquel Fernandes
Sara Camacho
 A) STOCKS AND SOLUTIONS

2 M CaCl2: HBS (2x HEPES buffered saline Plasmids for transfection:

pH 7.1, 500 mL):
- CaCl2.2H2O (MW=147.02 - GFP-Rev: 3.7 g/ l
g/mol) 29.4 g/100 mL - 50 mM HEPES - GFP Rev 1.4: 3.0 g/ l
- 280 mM NaCl - GFP: 1.8 g/ l
- 1.5 mM Na2HPO4

Drugs: Probe: PBS (10x pH 7,4):

- Leptomycin B: 10 g/mL - Hoescht (2 mg/mL) - 1,4 M NaCl

- Actinomycin D: 5 mg/mL - 27 mM KCl
- Cyclohexamide: 10 mg/mL - 100 mM Na2HPO4
- 180 mM KH2PO4

Lysis buffer (pH 7,5): Denaturing buffer Electrophoresis running buffer:

- 137.5 mM NaCl
- 100 mM Tris-Bicine - 100 mM Tris
- 5 mM EDTA
- 4% SDS - 100 mM Glycine
- 50 mM Tris-HCl pH 7.5 - 6 M urea - 0.2% SDS
- 0.5% Triton X-100
- 4% B-mercaptoethanol
- 10% Glycerol - Brilliant Blue
- CLAMP (1:1000)
- PSMF (1:000)

Transfer buffer(1L): TBS (pH 7.6, 10X, 1L): TBST:

- 3 g Tris (final concentration - 24.2 g Tris - TBS + 0.1% Tween 20

25 mM) - 80 g NaCl
- 14.4 g Glycine (final -
concentration 192 mM) TBST 5% skim milk (500 mL): TBST 0.5% skim milk (1000 mL)
- 200 mL Methanol (final
- 25 mg skim milk - 5 mg skim milk
concentration 20%)
- 500 mL TBST 1000 mL TBS

Primary antibodies: Secondary antibodies:

- Mouse anti-GFP antibody - Anti-mouse antibody

- Mouse anti-Tubulin - Anti-rabbit antibody
antibody
- Rabbit anti-Lamin A
antibody

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 B) PROCEDURE

a) FLUORESCENCE MICROSCOPY FOR CELL LOCALIZATION OF GFP-REV

1. Transfection of HEK293 cells using the calcium phosphate co-precipitation

method.
i. Take the HEK293 cells, which are cultured in coverslips in a 12 MultiWell
plate, and all the necessary solutions for the procedure into the laminar
flow chamber. (Note: Before placing something inside the chamber,
wash with ethanol in order to prevent contaminations.)
ii. Thaw the 2x HBS pH 7.1 solution at room temperature (RT). (Note: It is
not recommended to thaw the solution at 37 C).
iii. For each condition prepare 2 separate tubes, according to the table:

Table 1: Composition of the different transfection solutions used on the fluorescence

microscopy samples.

Volumes (for MW12)

ddH2O 427,4 l

A DNA 11,1 l
GFP
CaCl2 61,5 l

B HBS pH 7.1 500 l

ddH2O 433,1 l

A DNA 5,4 l
GFP-Rev
CaCl2 61,5 l

B HBS pH 7.1 500 l

ddH2O 431,8 l

A DNA 6,7 l
GFP-Rev1.4
CaCl2 61,5 l

B HBS pH 7.1 500 l

(Note: Add the CaCl2 solution dropwise)

iv. For each condition, add solution A (containing the DNA), dropwise, to
tube B, with the HBS buffer. Vortex for a few seconds.

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 v. Incubate the solution at RT for 30 min, vortexing for a few seconds
every 5 min.
vi. After the 30 min incubation, mix the solution again, and add the
preparations to the MW12 plate containing the cell culture (four wells
per plasmid). (Note: Add the mixtures to the medium dropwise).
vii. Gently rock the plate a few times to distribute the precipates in the
wells.
viii. Place the cells in the incubator (37oC, 5% CO2), for 4 hours.
ix. Remove the culture medium and wash the cells with 1 mL of HEK cell
medium.
x. To each well add 1 mL of fresh cell medium and place in the incubator
overnight (37oC, 5% CO2).

2. Cell treatment.
i. Treat the cells with the respective drugs, according to the scheme:

Table 2: representation of the treatments given to the cells.

A1 A2 A3 A4
Leptomycin B (4 l)
Cyclohexamide (1,5 l) +
GFP Control Cyclohexamide + Cyclohexamide (1,5 l)
(1,5 l) Actinomycin D (1 l) +
Actinomycin D (1 l)

B1 B2 B3 B4
Leptomycin B (4 l)
Cyclohexamide (1,5 l) +
REV-GFP Control Cyclohexamide + Cyclohexamide (1,5 l)
(1,5 l) Actinomycin D (1 l) +
Actinomycin D (1 l)

C1 C2 C3 C4
Leptomycin B (4 l)
Cyclohexamide (1,5 l) +
GFP-REV
Control Cyclohexamide + Cyclohexamide (1,5 l)
1.4
(1,5 l) Actinomycin D (1 l) +
Actinomycin D (1 l)

ii. Incubate for 3 hours.

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3. Fixation
i. Transfer the coverslips to a new 12 Multi-Well plate, with the cells facing
up, and incubate them with 4% Paraformaldehyde/4% sucrose solution
(500L per well) for 15 minutes, at room temperature.
ii. Transfer the coverslips to another 12 Multi-Well plate and wash the cells
twice with PBS (1mL per well).
iii. Incubate the cells with 100L of Hoescht reagent for each coverslip (diluted
1:2000), for 10 minutes, at room temperature, in order to stain the nuclei.
(Note: cover the coverslips with aluminum paper, because the Hoescht dye
is photosensitive).
iv. Wash the cells with PBS.
v. Mount the coverslips with DAKO mounting medium, with cells facing down,
towards the slide.
vi. Secure the coverslips on the slides by applying nail polish around them.
vii. Keep the slides covered from the light, at 4C.

4. Fluorescence Microscopy.
i. Using a fluorescence microscope, use an excitation wavelength of 350 nm
to excite the Hoescht probe and detect the position of the nuclei (emission
wavelength 510 nm) and take a picture.
ii. Excite the cells at 395 nm, without moving the cover glass, and detect the
emission at 470 nm and take a picture.
iii. Merge the two pictures and look for the GFP localization in cell: only in the
cytoplasm, in both the cytoplasm and nuclei or just in the nuclei.

B) WESTERN BLOT FOR TESTING PROTEIN EXPRESSION LEVELS

1. Transfection of HEK293 cells using the calcium phosphate co-precipitation

method.

i. Take the HEK293 cells, which are cultured in coverslips in a 6 MultiWell

plate, and all the necessary solutions for the procedure to the laminar flow
chamber. (Note: Before placing something inside the chamber, wash with
ethanol in order to prevent contaminations.)
ii. Thaw the 2x HBS pH 7.1 solution at room temperature (RT). (Note: It is not
recommended to thaw the solution at 37 C).
iii. For each condition prepare 2 separate tubes, according to the table:

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Table 3: Composition of the two mixtures used to transfect the cells for the Western blot
assay.

Volumes (for MW6)

ddH2O 866,2 l

A DNA 10,8 l
Rev
CaCl2 123,0 l

B HBS pH 7.1 1 ml

ddH2O 863,7 l

A DNA 10,8 l
Rev1.4
CaCl2 123,0 l

B HBS pH 7.1 1 ml

(Note: Add the CaCl2 solution dropwise)

iv. For each condition, add solution A (containing the DNA), dropwise, to tube
B, with the HBS buffer. Vortex for a few seconds.
v. Incubate the solution at RT for 30 min, vortexing for a few seconds every 5
min.
vi. After the 30 minutes incubation, mix the solution again, and add the
preparations to the MW6 plate containing the cell culture (three wells per
plasmid) (Note: Add the mixtures to the medium dropwise).
vii. Gently rock the plate a few times to distribute the precipates in the wells.
viii. Place the cells in the incubator (37oC, 5% CO2), for 4 hours.
ix. Remove the culture medium and wash the cells with 3 mL of HEK cell
medium.
x. To each well add 3 mL of fresh cell medium and place in the incubator
overnight (37oC, 5% CO2).

2. Cell treatment.
i. Treat the cells with the respective drugs, according to the scheme:

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 Table 4: representation of the treatments given to the cells for the Western blot assay

A1 A2 A3
Cyclohexamide (4,5 l) Cyclohexamide (4,5 l) +
REV Cyclohexamide + Actinomycin D (3 l) +
(4,5 l) Actinomycin D (3 l) Leptomycin B (12 l)

B1 B2 B3
Cyclohexamide Cyclohexamide (4,5 l) + Cyclohexamide (4,5 l) +
REV 1.4
(4,5 l) Actinomycin D (3 l) Actinomycin D (3 l) +
Leptomycin B (12 l)

ii. Incubate for 3 hours.

3. Cell extract preparation.

i. At the end of the incubation time, wash the cells 2x with 3 mL of PBS at
4C.
ii. Add the lysis buffer to the cells (180 per well).
iii. Incubate on ice during 15 minutes.
iv. Scrape the wells for approximately 1 min with a cell scraper
(Note: use one cell scraper for each condition).
v. Collect the samples from each condition to different tubes.
vi. Pipette up and down.
vii. Centrifuge the cells for 10 minutes at 3000 rpm at 4oC.
viii. Collect 100 l of the supernatant (cytoplasmatic fraction) in a tube
containing 25 l of the denaturing buffer.
ix. Discard the remaining supernatant.
x. Solubilise the pellet in 50 l of the lysis buffer.
xi. Centrifuge the preparation for 15 minutes at 3000 rpm at 4oC.
xii. Collect 50 l of the supernatant (nuclear fraction) to a tube containing
12 l of denaturing buffer.
xiii. Refrigerate the samples until ready to use.

4. Acrylamide gel preparation.

i. Assemble the 0.75 mm gel casting system and test for any leakages by
adding water.
ii. In a Falcon tube, prepare the 10 % acrylamide running gel according to
the table below (Note: for the purpose of this work, two gels were
made. Therefore, the volumes presented on the table below are relative
to the preparation of two gels.)

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 Table 5: Composition of the 10% acrylamide running gel.

10% Acrylamide Running Gel Volumes (for 2 gels)

ddH2O 5.6 mL

1.5 M Tris pH 8.8 4.6 mL

40% Acrylamide 3.5 mL

20% SDS 140 L

10% AMPS 140 L

TEMED 14 L

(Note: Acrylamide is toxic and should be pipetted using disposable pipettes)

iii. With a Pasteur pipette, fill up the gel casting system up to 1.5 cm from
the top.
iv. Add isopropanol on top of the gel (to exclude any air) and allow the gel
to polymerize.
v. After the polymerization of the running gel, prepare a 4% acrylamide
stacking gel, according to the following table:

Table 6: Composition of the 4% acrylamide stacking gel.

4% Acrylamide Stacking Gel Volumes (for 2 gels)

ddH2O 3.8 mL

0.625 M Tris pH 6.5 1.5 mL

40% Acrylamide 600 L

20% SDS 60 L

10% AMPS 60 L

TEMED 6 L

vi. Remove the isopropanol from the casting system, and fill it up to the
top with the stacking gel. Place the well divisors on the system and
allow the gels to polymerize. (Note: take care not to leave any air
bubbles on between the well divisor and the gel).
vii. After polymerization, remove the well divisors and insert the gels into
the electrophoresis apparatus. Fill the system with running buffer.

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5. SDS-PAGE.

i. Boil the sample + denaturing buffer mixture for 5 min.

ii. Charge wells with 25 L of the denatured samples. One well per gel
should be charged with 10 L of the molecular weight marker (the
NZYColour Protein Marker II was used).
iii. Run the SDS-PAGE at 120 V until the green band of the molecular
weight marker almost reaches the bottom of the gel.

6. Immunoblot.
(Note: for the purpose of the present work, the following procedure was done
twice, since two SDS-PAGE gels were used)

i. Cut a PVDF membrane the size of the SDS-PAGE gel, and two pieces of
filter paper slightly larger.
ii. To activate the PVDF membrane, place it in methanol for a few seconds
and then equilibrate it in transfer buffer.
iii. Set up the transference cassette according to the following order:

Cathode facing cassette panel (with corresponding sponge)

Filter paper
SDS-PAGE gel
Activated PVDF membrane
Filter paper
Anode facing cassette panel (with corresponding sponge)

iv. Run the electrotranference at 20 mA overnight, at RT.

v. After the electrotransference is finished, block the PVDF membrane
with TBS-T 5% skim milk solution for 1 hour.
vi. Incubate the membrane with the mouse anti-GFP and anti-Tubulin and
with the rabbit anti-Lamin A primary antibodies diluted in the TBS-T
0.5% skim milk solution for 1h (at RT)
vii. Wash the membrane 5x for 3 min with the TBS-T 0.5% skim milk
solution.
viii. Incubate the membrane with an anti-mouse and with an anti-rabbit
secondary antibodies, coupled to alkaline phosphatase for 1h (at RT).
ix. Repeat step vii.

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x. Spread 500 L of the ECF substrate on a Petri dish, and lay the
membrane on top of the substrate with the proteins facing down.
Incubate for 5 min (at RT).
xi. Use a detection system to detect the fluorescence signals on the
membrane.

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