Nu page xcell surelock module

Immunoblot was performed following a previous published protocol.^{6 (link)} MCF-7 cells were treated at different concentrations of AmbI (0.008, 0.016, 0.4, 2.0 and 10 μM) for 3 h. The cells were lysed using PhosphoSafe Lysis Buffer (Novagen), and the lysates were analyzed by western blot analysis with primary (1:1000) and secondary antibodies (1:2000). Protein concentration in the lysate was determined using a Bradford protein assay kit with an albumin standard (Thermo Scientific). Absorbance was measured using Fluostar Optima plate reader (BMG Labtech Inc, Durham, NC). Equal amounts of protein (20 μg) were loaded together with LDS sample loading buffer (Invitrogen) and resolved using Nu-PAGE 10% SDS-PAGE Bis-Tris gels together with SeeBlue® Plus2 Pre-Stained Standard (Invitrogen).
Electrophoresis was performed using SDS-PAGE buffer in a Nu-PAGE XCell SureLock Module from Invitrogen. Proteins were transferred to a polyvinyldiene fluoride (PVDF) membrane using transfer buffer. The blots were blocked at room temperature using non-fat milk and probed using primary antibodies against each target protein using BSA in TBS-T overnight. Conjugated antibodies were detected using Chemiluminescent substrates Supersignal Femto kit from Thermo Scientific and relative band densities were determined.

To confirm the effect of a compound on the NF-κB pathway, cells were treated at different concentrations (0.008, 0.016, 0.4, 2.0 and 10 μM). Briefly, cells were lysed with PhosphoSafe Lysis Buffer (Novagen) and lysates analyzed by western blot analysis with primary (1:1000) and secondary antibodies (1:2000). Protein concentration in the lysates was determined by using the Bradford protein assay kit and an albumin standard (Thermo Scientific). Absorbance was measured using the Fluostar Optima plate reader (BMG Labtech Inc, Durham, NC). Equal amounts of protein (20 μg) were loaded together with LDS sample loading buffer (Invitrogen) and resolved using Nu-PAGE 10% SDS-PAGE Bis-Tris gels together with SeeBlue^® Plus2 Pre-Stained Standard (Invitrogen). Electrophoresis was performed using SDS-PAGE running buffer in a Nu-PAGE X Cell SureLock Module from Invitrogen. Proteins were transferred to a polyvinyldiene fluoride (PVDF) membrane using transfer buffer, TBS-T. The blots were then blocked at room temperature using non-fat milk and probed using primary antibodies against each target protein using BSA in TBS-T overnight. Conjugated antibodies were detected using the chemiluminescent substrate Supersignal Femto kit from Thermo Scientific and relative band densities were determined.