Proteoextract subcellular proteome extraction kit s pek

Five million HEK293T cells were transfected with 5 μg VR1012, hBCA2, or fBCA2. Forty-eight hours later, the cells were lysed and proteins were extracted to obtain cytoplasmic and nuclear extracts using the ProteoExtract^® Subcellular Proteome Extraction Kit (S-PEK), following the manufacturer’s instructions (Millipore, Bedford, MA, United States). The cytoplasmic fraction and nucleic protein fraction were analyzed by western blotting. Equal protein loading for cytoplasmic and nuclear fractions was confirmed by blotting against GAPDH.

Five million (5 × 10⁶) 293T or HeLa cells were transfected with 5,000 ng of either an empty vector (pcDNA5) or a vector encoding HA-BCA2 (pcDNA5-HA-BCA2) or a BCA2 mutant (pcDNA5-HA-A₂₂₈A₂₃₁). Forty-eight hours later, the cells were lysed, and sequential extraction of proteins from specific compartments and/or organelles within the cell was performed using a ProteoExtract subcellular proteome extraction kit (S-PEK) (Millipore, Bedford, MA), following the manufacturer's instructions. The cytoplasmic fraction (F1) and nucleic protein fraction (F3) were analyzed by Western blotting using HA-, IκBα-, p100/p50-, and RELA/p65-specific antibodies (Table 1). Equal protein loading for the cytoplasmic and nuclear fractions was confirmed by blotting against β-actin (a cytoplasmic and nuclear marker). The purity of the nuclear and cytoplasmic fractions was determined by detecting lamin A/C (a nuclear marker) and LAMP-1 (a cytoplasmic marker), respectively.