BT-474 and HCC1419 cell lines were obtained from American Type Culture Collection and cultivated for no more than 2 months after each frozen aliquot was thawed. Amphotropic retrovirus carrying Taspase1 specific knockdown hairpin was generated as described26 (link),29 (link). To assay cell proliferation, 1 × 105 cells were seeded onto each well of a 6-well plate and counted 4 days later. Cell cycle and cell death analyses were performed as described26 (link). For western blot, cells and tissues were lysed in standard RIPA buffer. The anti-Taspase1 rabbit polyclonal antibody is as described22 (link),26 (link). Antibodies for cyclin E2 (4132, Cell Signaling), cyclin A (C4710, Sigma), p21 (sc-397, Santa Cruz Biotechnology), p27(sc-528, Santa Cruz Biotechnology), cyclin D1(sc-450, Santa Cruz Biotechnology), p16(554079, BD Pharmingen), and ErbB2(OP-15, Calbiochem) were purchased from indicated companies. Antibodies were detected using the enhanced chemiluminescence method (Western Lightning, PerkinElmer). Immunoblot signals were acquired with the LAS-3000 Imaging system (FujiFilm) and were analyzed with ImageJ software.
Protocol detail
Taspase1 Knockdown in Breast Cancer Cell Lines
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BT-474
HCC1419
cyclin A (C4710,
p21 (sc-397,
p27(sc-528,
cyclin D1(sc-450,
Western Lightning
LAS-3000 Imaging system
Anti antibody
Antibodies
Assay
Buffer
Cell cycle
Cell death
Cell lines
Cell proliferation
Cells
Chemiluminescence
Cyclin a
Cyclin d1
Cyclin e2
Erbb2
Frozen
Immunoblot
Rabbit
Retrovirus
Ripa
Tissues
Western blot
Corresponding Organization : Memorial Sloan Kettering Cancer Center
Other organizations : Washington University in St. Louis
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Variable analysis
independent variables
- Taspase1 specific knockdown hairpin
- Cell proliferation
- Cell cycle
- Cell death
- Protein expression (Taspase1, cyclin E2, cyclin A, p21, p27, cyclin D1, p16, ErbB2)
- Cell lines (BT-474 and HCC1419)
- Cultivation duration (no more than 2 months after each frozen aliquot was thawed)
- Not explicitly mentioned
- Not explicitly mentioned
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