I didn't understand your question, but if you don't see the protein in your membrane could be your extraction process or your antibody. You need a positive control to be sure that you are transferring the proteins. We transfer our membranes 1h, 100V. Make sure that you activate your membrane 10 min with metanol.

I suppose GFP is the reporter of your plasmid that been drove simultaneously with your gene of interest, that's why you made such statement. Since GFP signal was observed in 293T, then the problem lies in WB, but there's not enough details for troubleshooting, for a positive signal, you have to employ a positive control as suggested by Yolanda, and you might also want to share how you processed the protein.

Many thanks for the suggestion. Actually, I can see signal for my positive control in WB. So, the problem should be from expression. I again check the pMSCV vector and notice: PGK promotor was just in front of Puro-R and GFP, but there's no promotor before my target gene. So I doubt:

(1) Due to PGK promotor, Puro-R and GFP can be overexpressed in transit transfection.

(2) But for my target gene, (cloned between XhoI/EcoRI). It will not be overexpressed in transit transfection, due to lack of promotor.

(3) The expression of my target will follow the step below:

(a) Transfection of 293 cell and collection of virus particle.

(b) Infection of cell with virus particle.

(c) The target gene integrate into genome of the transfected cell.

(d) Overexpression of the target gene by the cell.

Please help to check whether my hypothesis iscorrect.

if "there's no promotor before my target gene"... then you have the answer