Silencing of GSK-3 isoforms in 22Rv1 cells. (A) 22Rv1 cells were transfected with the indicated shRNAs and then selected with puromycin, as described in the Methods. After 72 hours, total cell extracts (20 μg) were prepared and probed by western blotting using the indicated phosphospecific antibodies and then stripped and reprobed with a mixture of antibodies to GSK-3α, GSK-3β and HSP60 (top panel). (B) Expression of GSK-3 normalized to HSP60. (C-E) Cells were co-transfected with a combination of eGFP and the pSM2 plasmids αsh1 and αsh2 (C), βsh1 and βsh2 (D) or αsh2 and βsh2 (E). 72 hours after transfection, cells were fixed and stained with anti-GSK-3α or anti-GSK-3β monoclonal antibodies and analyzed by confocal microscopy. Arrowheads indicate GFP+ cells in which GSK-3α or GSK-3β are silenced and asterisks indicate GFP+ cells with the same level of protein expression as neighbouring cells. Scale bars = 50 μm.