Anti s6 ribosomalprotein antibodies

Fibroblasts grown from normal skin and skin tumors obtained during the treatment
period were plated on 100-mm dishes and were incubated in Dulbecco’s modified
Eagle’s medium with or without 10% fetal bovine serum, with or without 2
nM sirolimus for 24 hours. Cells were lysed with protein extraction buffer, separated on
10% Mini-PROTEAN^® precast polyacrylamide gels, transferred to
polyvinylidene fluoride membranes and immunoblotted with β-actin (Sigma-Aldrich),
anti-TSC2 (D93F12), anti-phospho-S6 ribosomal protein (Ser-235/236) and anti-S6 ribosomal
protein antibodies (Cell Signaling Technology) as described in previous methods.
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CLL cells were incubated in microtubes with phosphate-buffered saline (PBS) supplemented with 2% bovine serum (HyClone) and with anti–S6 ribosomal protein antibodies (Cell Signaling Technology) and anti-CD5 antibodies (Becton, Dickinson and Company) for 1 hour. After being washed three times with phosphate-buffered saline, the cells were suspended in 5 mg/ml solution of 4′,6-diamidino-2-phenylindole (DAPI) dye (Invitrogen) for 5 minutes and then washed in phosphate-buffered saline to remove the unbound dye. The cells were then placed into μ-slide VI^0.4 chamber slides (ibidi, LLC) for microscopic analysis. The slides were viewed using an Olympus FluoView 500 confocal laser scanning microscope (Olympus America), and images were analyzed using the FluoView software (Olympus America).