TITLE: CagA phosphorylation assay and its semiquantitative analysis
AUTHORS: Anna F. Zeitler, Luisa F Jimenez-Soto

[DESCRIPTION]
CagA is the oncogenic toxic of Helicobacter pylori. The toxin is injected into host cells and tyrosine phosphorylated in the C-terminal EPIYA motifs by host kinases. Positive translocation of the CagA toxin into the cell is evaluated by detecting the phosphorylated form in the cell lysate using antibodies detecting phosphotyrosines. Previously detection was limited to a positive o negative results, but with new advances in semiquantitative western blots we are able to quantify changes on CagA translocation / Phosphorylation and get a better idea of the dynamics of injection.
The protocol described here has been used for evaluation of CagA phosphorylation and its semiquantitative analysis in the following publications:
DOI:10.1111/cmi.12166; DOI:10.1038/srep23227,  and DOI:10.1016/j.mimet.2016.04.016

[BEFORE_START]
Make sure you know how to use all equipment in the lab, and read the safety warning of all chemicals used before you use them.

[GUIDELINES]
Make sure you follow the biosafety protocols and recommendations of your laboratory.


[STEPS]
SECTION: Seed AGS cells 48 hours before infection will be performed.
2. The objective is to have a cellular confluency in the 6-well of 80-90% at the moment of infection. For you to achieve this, you have to know your cells and their multiplication speed. For AGS cells the usual splitting for 48 hours with synchronization (see below) was 1:8  (from a 90% - 100% confluent culture)
SECTION: Prepare H. pylori bacteria for infection
1. H. pylori has a amazing capacity to change during growth. In order to keep the experiments reproducible and comparable, use relative fresh bacteria for all infection experiments.
On Friday afternoon, take from frozen stocks the bacteria by scratching the surface of the ice with the inoculation loop. A piece of "ice" will adhere to the loop. Do not distribute the material on the plate. Just place it on the agar and let H. pylori grow on the small drop of defrosted media, undisturbed. Place the agar plates to grow in an incubator with microaerophil conditions (10% CO2, 5% O2 and 75% N2) at 37°C for the weekend.
On Monday, passage the bacteria to a new GC Cholesterol plate 
 
, and pass them onto a new plate on Tuesday for Wednesday's work, where bacteria are fit enough to be used for infection. 
 

 
SECTION: Synchronize the AGS cells 16h before infection
3. Remove serum-containing media (RPMI + 10% Inactivated FCS, Complete Media (CM)) and change it for media without serum (RPMI without serum) around 16 hours before infection, in order to synchronize cells in G0.
 
SECTION: Resuspend bacteria
4. For 1/4 of plate grown bacteria, collect all bacteria using a sterile cotton swab and introduce it in a tube containing at least 500 µl of PBS. Using circular movements, remove the bacteria culture attached to the cotton fibers using the PBS solution. The objective is to have as much bacteria in the solution, and reduce the amount of liquid attached to the cotton swab before discarding it.
SECTION: Measure the optical density (OD) of the bacterial suspension at 550 nm
5. Make a 1:100 dilution of bacteria suspension in PBS (10µl bacterial suspension in 990µl PBS) and measure its optical density (OD) in a densitometer at 550 nm.
SECTION: Calculate the amount of H. pylori colony forming units (CFU) per ml of suspension
6. Multiply the dilution factor to obtain the OD550 / ml of suspension.
Calculate the amount of bacteria in your suspension taking into consideration that and OD550 of 0,1 is equivalent to aprox. 3x107 CFU/ml. 
 
SECTION: Infect your cells with an Multiplicity of Infection (MOI) of 60.
7. Multiplicity of Infection (MOI) of 60 means that you will add 60 bacteria for each cell in the well. For general calculations, each well of a 6-well cell culture plate has approx. 1x106 adherent cells at a confluency of 90% to 100%
SECTION: Prepare the AGS cells 30 min before infection
8. Remove the serum-free media from the cells and add 1 ml of complete media (CM) per well around 30 min before infection
SECTION: Infect the cells
9. Infect cells and incubate at 5% CO2 37°C for 3 to 4 hours.
 
SECTION: Quantify signals relative to control infection
24. Using the software ImageLab from Bio-Rad, set lanes and bands. Quantify signals using relative values to the control infection.
 

SECTION: Stop infection for harvestinfection
10. Stop the infection by placing the plate on ice. For the rest of the procedure, maintain the samples cold.
 

SECTION: Collect supernatant for cytokine measurements
11. Collect the media for cytokine measurements. If no cytokine measurements are necessary, remove the media with help of a vacuum pump. Add 1 ml PBS* (PBS with protease and phosphatase inhibitors). Prevent the well’s surface from drying out (crystal formation damages the cells).
 
SECTION: Detach cells
12. With a cell scrapper detach the cells from the bottom of the plate. 
 

SECTION: Collect cells
13. Collect the cells suspension in 1,5 ml tubes. Centrifuge the cells 500 g for 10 minutes at 4°C in a swing rotor centrifuge.
SECTION: Prepare samples for Western blot
14. Discard ALL the supernatant taking care of not loosing the pellet. 
Resuspend the pellet in 20 µl of your favorite Lysis buffer containing protease inhibitors.
Add immediately 25µl of 2X SDS loading buffer and boil the samples at 95°C for 10 min. To avoid condensation and stickiness of DNA, place the tubes immediately in ice. Do not centrifuge!

 

SECTION: Separation of proteins
15. For separation of proteins, we use the protocol Single Gel system based in Ahn T et al (2001)
SECTION: Western blot - Block the membranes
18. Block membrane with TBS-T (TBS + 0,075% Tween 20) with 3%BSA for 1 hour at room temperature.
SECTION: Western blot - Primary antibody
19. Add the anti-phosphotyrosine antibody in a dilution 1:10000 dilution (Clone 4G10 antibody  from Millipore), for 1 hour in PBS with 3% BSA at room temperature.
SECTION: Western blot - Remove excess primary antibody
20. Wash 4 times, 15 minutes each, with 5 ml TBS-Tween (TBS + 0,075% Tween 20) at room temperature.
SECTION: Western Blot- Secondary antibody
21. Add 1:10000 anti-mouse peroxidase conjugated antibody (Sigma) for 45 minutes  in TBS 0,075% Tween 20 .
SECTION: Western blot- Removal of excess secondary antibody
22. Wash 4 times 15 minutes with 5 ml TBS 0,075% Tween 20 at room temperature.
SECTION: Western bot - Detection
23. Remove ALL WASH BUFFER . Take care to not to let the membrane dry in this step!!!
Add Millipore Immobilon Western (Cat Nr. WBKLS0500) developing solution (1 ml solution A, 1 ml solution B).
Let the membrane roll with the developing solution for about 1 to 2 min
Place membrane between to two transparencies.
Place membrane in Gel documentation machine (Gel Doc™ XR+ Gel Documentation System) and detect the chemoluminiscence
SECTION: Activate PVDF membranes
17. Place the membranes in contact with methanol to activate them.
 

 

SECTION: Transfer of proteins to PVDF membrane
16. Activate gel for detection of proteins using the stainfree system for quantification of proteins present in the sample. These values allow us to do the normalization of the phosphorylation signals after immunodetection.
We use the Bio-Rad Gel Doc™ XR+ Gel Documentation System for the stainfree detection.
For transfer of proteins to the PVDF membrane, we use the protocol Trans-Blot® Turbo™ Transfer with Home-made buffers
SECTION: Normalize values
25. Using the same image and the image of the Stainfree gel, use the normalization option from the ImageLab software. Multiply the values obtained for the phosphorylation signal with the values obtained for the normalization.