CHAPTER 1; INTRODUCTION
PHOSPHORYLATION UPON STIMULATION OF LUNG FIBROBLAST CELLS WITH ET-1:
2.6.1: IMMUNOPRECIPITATION OF CELLULAR PHOSPHOPROTEINS:
i) Stimulation:
Cells were incubated overnight in 5ml of serum starvation medium (37°C, 5% CO2).
The purpose of this was to reduce the normal levels of cell stimulation from the medium, heightening the impact of subsequent stimulation with ET-1.
Serum starvation medium:
MEM containing: 200mM L-Glutamine 200mM NEAA 0.5% FBS
The following day, the cells were divided into two groups. 250p-l of IpM ET-1 in PBS was added to one group (a final concentration of 50nM), henceforth referred to as the ‘Stimulated’ group, which was incubated for a stimulation period of 2 mins at 37°C, then transferred immediately to ice. 250p,l of PBS was added as a control to the other group - ‘Unstimulated’, which was also incubated for 2 mins at 37°C and then
transferred to ice. The medium was aspirated from both groups, and the cells were washed twice with 5ml of ice cold PBS/PI
ii) Sample Preparation:
Cells were scraped on ice in 0.5ml/dish of ice-cold lyses buffer (MAP), and sonicated for 3 X 10 0.5s bursts. The samples were spun down at 13000 rpm for 20 mins at 4°C.
The pellet was discarded, and the supernatant, containing solubilised cellular proteins, made up to 0.2M NaCl then incubated, rotating, with 200p.l of 50% Sepharose 4B at 4°C.
After 1 hour the mixture was spun down for 2 mins at 3000 rpm, 4°C to remove the sepharose and any associated proteins. A solution of 1% low m.p. agarose was added to the supernatant to give a final concentration of 0.05% agarose, and the sample was vortexed briefly. The sample was spun down again at 3000 rpm, 4°C for 2 mins to remove the agarose. Some common cell proteins, including many nuclear proteins and transcription factors have some affinity for agarose, and the incubations with sepharose and agarose were performed in order to reduce non-specific binding to the agarose used as a carrier for the phospho-antibodies.
The supernatant was incubated for 3 hours at 4°C with 250p,l of 50% anti- phosphoserine-agarose on a rotator.
Empty micro-columns (Mobitec (Gottingen, Germany)) were loaded with a maximum of 700p,l of sample and spun briefly to about 5000 rpm. Columns were washed 3 times
in rapid succession with 0.5ml of cold lysis buffer (MAP), then once with 0.5ml of ice- cold lOmM Tris-HCl pH 7.4. The agarose was immediately resuspended in ice cold EEF buffer, modified by leaving out the DTT, to give a total volume of 300p,l per gel to be run, and incubated at RT for 1 hour to elute the proteins from the agarose. The DTT is omitted in order to prevent unnecessary breakdown of the IgG by DTT, which leads to increased numbers of background spots on the gel. Columns were loaded and washed no more than 4 at a time as rapid washing is important due to the low affinity of the antibodies for phosphoproteins.
After 1 hour of incubation at RT, the samples were made up to 65mM DTT. lEF strips (Amersham Pharmacia Immobiline Drystrips, pH 3-10, linear gradient, 13cm) were reswelled in 300p.l per strip of sample overnight, with a layer of mineral oil over the top to prevent dehydration.
Immunoprécipitation of tyrosine and threonine phosphorylated proteins was done in the same way as described above, but with anti-phosphotyrosine-agarose or anti- phosphothreonine-agarose.
iii) Isoelectric focussing:
Isoelectric focussing is a form of electrophoresis that separates proteins on the basis of their isoelectric points. Strips of polyacrylamide gel, incorporating an immobilised pH gradient, are loaded with sample as described above. They are then subjected to a series of focussing steps under gradually increasing voltages, which cause charged proteins to move along the gradient and concentrate at their isoelectric points. The exact series of
voltage steps to be used for focussing depends on the range of the gradient used, the length of the gel strip, and the content of the sample.
In this case, the following protocol was used on an Amersham IPGPhor lEF system: Strips were transferred to IPGPhor strip-holders, and covered with 2ml of mineral oil to prevent drying. Focussing was carried out as follows:
Step Voltage (V) V olthours (Vhr)
I 30 30 2 300 150 3 1000 500 4 3000 3000 5 8000 60,000 6 2000 24 hours
This is a ‘step-and-hold’ protocol, which means that after the allotted number of Volthours, the voltage is moved up to the next step in one go, and remains constant until the target number of Volthours is reached, rather than increasing gradually at a constant rate. The IFF process was carried out at I6°C, and the maximum current per strip was 50pA. The purpose of step 6 is to give the operator some leeway in choosing when to end the focussing (i.e. to allow the protocol to run overnight). A voltage of 2000V maintains the focussing by preventing proteins from diffusing away from their isoelectric points, but does not cause ‘over focussing’, as would occur if the strips were left at 8000V for this time.
The IFF strips were equilibrated prior to 2°^ dimension electrophoresis. The mineral oil was washed off with ddHzO, and the strips were incubated at RT in trays with an
individual slot for each strip, face up on a shaker, firstly in 2ml O.IM DTT in equilibration buffer for 20 mins, then in 2ml 0.2M iodoacetamide in equilibration buffer for 20 mins.
iv) 2°^ Dimension gel electrophoresis:
The procedures for lEF strip loading and 2"^ dimension gel electrophoresis are described in section 2.2.2. The protein separation was visualised by silver staining the gels, as described in section 2.2.3.
v) Analvsis bv mass spectrometrv:
Gels were scanned using a UMAX Power Look m scanner at a resolution of 800 dpi. The images were analysed by eye, and also using Melanie 3 software, to look for features that appeared in ‘Stimulated’ or ‘Unstimulated’ gels, but not both.
Selected spots were cut out with a clean scalpel, and digested in the gel using trypsin, as described in section 2.2.5. The digests were subjected to MALDI-TOF analysis to identify the proteins by mass fingerprinting. Where the result was uncertain, the most likely possibilities were further investigated by nanospray sequencing. Procedures for these methods are described in section 2.2.5.