Docstoc
EXCLUSIVE OFFER FOR DOCSTOC USERS
Try the all-new QuickBooks Online for FREE.  No credit card required.

Indirect immunofluorescence and confocal microscopy

Document Sample
Indirect immunofluorescence and confocal microscopy Powered By Docstoc
					                       SUPPLEMENTARY INFORMATION

Materials and Methods



Indirect immunofluorescence and Confocal laser scanning microscopy



1.5 x 106 Jurkat Puro or Jurkat Bcl-2 cells were plated in complete growth medium in

the presence or absence of 1.0 μΜ 2-ME2 for 24 h. The cells were then centrifuged,

resuspended in ice-cold PBS containing Ca2+ and Mg2+ and placed on coverslips

pretreated with 0.01% alcian blue in sterile distilled water for 30 min to attach.

Following fixation and permeabilization, the cells were incubated with antibodies to

Bcl-2, p27Kip1, p50 or p65 at 1:50 dilution in 10% FCS/PBS for 45 min at room

temperature followed by secondary antibodies conjugated with FITC or TRTC

(Dianova, Germany) at 1:200 for 1 h at RT.

  To visualize the nuclei, the stained cells were treated with RNase A (1μg/ml) for

30 min, washed with PBS and propidium iodide-TRITC was added and incubated for

10 min. The stained cells were washed with PBS and mounted on glass slides.

Excitation of FITC and TRITC was achieved using the 488 and 568 nm wavelengths,

respectively. Images were collected on a Leica TCS-SP scanning confocal

microscope, equipped with an argon/krypton laser and Leica TCS software.



References

Papanikolaou A, Papafotika A, Murphy C, Papamarcaki T, Tsolas O, Drab M, et al.

Cholesterol-dependent lipid assemblies regulate the activity of the ecto-

nucleotidase CD39. J Biol Chem 2005;280:26406-26414.




                                         1
                   LEGENDS TO SUPPLEMENTARY FIGURES



Fig. S1 - Subcellular localization of Bcl-2 and p27Kip1 in Puro- or Bcl-2-expressing

Jurkat cells following 2-ME2 treatment. Jurkat Puro and Jurkat Bcl-2 cells were

attached onto coverslips using alcian blue, fixed, and stained with a monoclonal anti-

Bcl-2 (A) or a monoclonal anti-p27Kip1 antibody (B) and analyzed by confocal laser

scanning microscopy to determine the subcellular localization of Bcl-2 and p27Kip1 in

the presence and absence of 2-ME2.



Fig. S2 - Subcellular localization of p65 and p50 in Puro- or Bcl-2-expressing Jurkat

cells following 2-ME2 treatment. Jurkat Puro and Jurkat Bcl-2 cells were stained with

an anti-p65 or anti-p50 antibody and analyzed by confocal laser scanning

microscopy to determine the subcellular localization of NF-κB subunits in the

presence and absence of 2-ME2. Note (i) that in contrast to Jurkat Bcl-2, in Jurkat

Puro cell cultures only a few cells survived 2-ME2 treatment and (ii) the higher

intensity and sharpness of the staining in Jurkat Bcl-2 compared to Jurkat Puro cells.




                                          2

				
DOCUMENT INFO