(1) I am using a cell line expressing STAT1-eGFP to demonstrate the nuclear translocation of STAT1-eGFP after IFN-treatment.
My purpose is to compare the efficacy of IFNs with this system.
Briefly, HEK293 (or HELA) cells were transfected with a plasmid STAT1-eGFP. (The plasmid should be fine because it was bought from Addgene, and other published studies have used the same plasmid for the same nuclear translocation experiment.)
I have transitorily transfected cells, and I also have HEK293 stably expressing STAT1-eGFP.
STAT1-eGFP located in cytoplasm that is consistent with others’ researches.
Then I treated those cells with 1000U/ml IFNbeta or IFNalpha. However, I have not observed the accumulation of STAT1-eGFP in nuclear after 30min, 1hours, 2hours, 4hours, 8hours, and 12hours post-treatment.
IFNs should be fine. I have used them for IFN-receptor internalisation experiment. They worked very well.
I do not understand why there was no nuclear trafficking.
(2) I have also used immunofluorescence to detect the phosphorylation state of STAT1.
Firstly, HELA (HEK293, Raji and U973) was cultured on slide, then cultured in medium with 1% serum 24 hours before IFN-treatment. 1 hour before IFN treatment, cells were cultured in medium without serum.
After IFN-treatment, cells was washed, fixed and permeabilized (I have tried Paraformaldehyde 4% for fixation then 1% saponin; or cold methanol treated at -20C for 10min). After washing, rabbit anti-STAT1-p (T701) (from Cell signalling) was used as 1st antibody, then goat anti rabbit antibody-Alexa594 (I have used whole goat IgG and also its F(ab)’2; from Cell signalling) was used as 2nd antibody.
However, I did not detect any specific signalling of STAT1-p.
The last possibility that I will check is the serum for cell culture medium. My colleagues also have a bit problem with this serum when doing cytokine related experiment. However, I have starved cells before IFN-treatment. I am very confused.
Any suggestion and help is appreciated!
Thanks a lot!
Fresa-niMember Since 25 Jan 2013
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29 Jan 2013 - 06:15