To confirm these results, pooled cells transfected with VerUTR or the control vector were lysed and probed with anti-PTEN antibody for western blot analysis.
Cells of one 150 mm dish transfected with pFLAF-NKX3.1 or empty vector were lysed in each 1 ml IP lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X 100) on ice.
The transfected cells with either empty vector or 3 x Flag-tagged Gls2 expressing vector were lysed in M2 lysis buffer (20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM glycerol phosphate, 1 mM sodium vanadate and proteinase inhibitor cocktail).
Cells transfected with lentiviral vectors were lysed with lysis buffer at 4°C for 30 min.
HEK and PC12 cells transfected with the empty vector or S. oneidensis or E. coli NMN deamidase (WT and mutant) expressing vectors were lysed by sonication in 50 mM Tris HCl, 150 mM NaCl, 1 mM DTT and protease inhibitors.
Transfected HEK 293 cells with appropriate expression vectors were lysed in a lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 1.5% aprotinin, and 1 mM phenylmethylsulfonyl fluoride.
For immunoprecipitations, HeLa cells transiently transfected with FLAG-tagged ubiquitin vectors were lysed in lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 5 mM EDTA).
Cells transfected with the desired expression vectors were lysed in ELB buffer (50 mM Tris/HCl pH 7,5; 120 mM NaCl; 20 mM NaF; 1 mM EDTA; 6 mM EGTA; 15 mM sodium pyrophosphate; 1 mM phenylmethylsulfonyl fluoride; 0,2% NP-40; 10% glycerol and a protease inhibitor mix containing Aprotinin, Leupeptin and Pepstatin).
Briefly, a 100 mm tissue culture dish of Cos-7 cells, transfected with each of the scFvE-cyto recombinant vectors, was lysed with 300 μl ice-cold extraction buffer containing containing Tris-Cl 20 mM, pH 8, MgCl2 20 mM, 0.5% NP40, 0.1 mg/ml leupeptin, chymostatin, and aprotinin, and 0.1 mM PMSF for 15 minutes.
After 30 h of transfection with each Tax vector or control vector, cells were lysed, and total RNA was prepared using the RNeasy Mini Kit (Qiagen).
After 30 h of transfection with each Tax vector or control vector, cells were lysed, separated through a 6 10% sodium dodecyl (SDS -polyacrylamide gel, and then tranSDS -polyacrylamideembrane (Immobilon-P, Millipore Corp).
