Mouse anti β1 integrin antibody p2d5

β1-integrin of W1 and W1CR cells was knocked down using viral transduction. First, cells were seeded in a 96-well plate at a density of 1000 cells per well and incubated overnight in RPMI medium containing additives. The next day, the medium was substituted by 100 µL per well of transduction medium (Polybrene^® 4 μg/mL in complete cell medium). Also, the viral particles (control shRNA lentiviral particles-A and ITGB1 shRNA (h) lentiviral particle: sc-35674-V; Santa Cruz Biotechnology, Heidelberg, Germany) were thawed and resuspended. In one well, 4 μL of integrin β1 shRNA (h) lentiviral particles were added to the medium, the same procedure was carried out for the control shRNA lentiviral particles in another well. One day later, the media was changed, and the still infectious medium containing lentiviral particles were used to transfect another well in the same manner. The cells were incubated in a medium containing 2.75 µg/mL puromycin (Carl Roth GmbH) to enable the selection of stable clones. The transfected cells were further cultured until they nearly reached confluence and then passaged for further experiments. Knockdown was confirmed by Western blot using a mouse anti-β1-integrin antibody [P2D5] (Santa Cruz Biotechnology).

ITGB1 of W1 and W1CR cells was knocked down using viral transduction as described before [9 (link)]. Cells were seeded in a 96-well plate at a density of 1000 cells per well and incubated overnight in RPMI. The next day, the medium was substituted with 100 µL per well of transduction medium (Polybrene^® 4μg/mL in complete cell medium). Also the viral particles (control shRNA lentiviral particles-A and ITGB1 shRNA (h) lentiviral particle: sc-35674-V; Santa Cruz Biotechnology, Heidelberg, Germany) were thawed and resuspended. In one well, 4 μL of ITGB1 shRNA (h) lentiviral particles were added to the medium, the same procedure was carried out for the control shRNA lentiviral particles in another well. After one day, media were changed and medium containing the still infectious viral particles were used to transfect another well in the same manner. For selection of stable clones, cells were incubated in medium containing 2.75 µg/mL puromycin (Carl Roth GmbH, Karlsruhe, Germany). Knockdown was confirmed by Western blot using a mouse anti-β1-integrin antibody [P2D5] (Santa Cruz Biotechnology).

β1-Integrin of MCF-7 cells was knocked down by using viral transduction. Cells were seeded at a concentration of approximately 1000 cells per well of a 96-well plate and incubated overnight in DMEM medium containing additives. The next morning, polybrene was added at a concentration of 5 µg/100 µL and 12 µL of β1-integrin or scrambled control shRNA (h) lentiviral particles were added according to manufactures protocol (Santa Cruz Biotechnology) and calculated from the recommended lentivirus MOI (multiplicity of infection) for MCF-7cells. The next day, media was changed and the still infectious medium containing lentiviral particles (Santa Cruz Biotechnology) were used to transfect another well in the same manner. The transfected cells were further incubated in fresh medium containing 0.3 µg/mL puromycin (Carl Roth GmbH) until they nearly reached confluence and then passaged for further experiments. Knock-down was confirmed by Western blot using a mouse anti-β1-integrin antibody (P2D5, Santa Cruz).