Phosphatase halt

Cells were seeded at 500,000/ml and incubated as indicated. Proteins were extracted using RIPA (50mM Tris-HCL ph8.0, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS), 7x phosphatase inhibitor (Roche #04693159001), Phosphatase Halt (Thermo #78428) and Triton X-100. Proteins were quantified using the BCA Assay (Thermo #23225) with 30μg loaded per lane for western blotting. All blots were developed using autoradiography film (GeneMate #F-9023-8x10) after incubation with antibodies indicated above.

Cells were seeded at 500,000/mL and incubated as indicated. Proteins were extracted using RIPA (VWR, Radnor, PA, USA), Phosphatase Halt (Thermo #78428). Proteins were quantified using the BCA assay (Thermofisher Scientific, Waltham, MA, USA) with 20 ug loaded per lane for Western blotting. All blots were developed using autoradiography film (VWR, Radnor, PA, USA) or Li-Cor Odyssey Fc imaging system after incubation with antibodies indicated above. Densitometric analysis conducted using Li-Cor affiliated ImageStudio software, with all analyses normalized to loading controls. All antibodies were used per the manufacturer recommended dilutions.