Western blotting analysis was used to semiquantitatively determine LMP2A, TWIST, YB-1 and HER2 protein levels. The primary antibodies used were anti-LMP2A (a gift from Prof. Mu-sheng Zeng), anti-TWIST (H81; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-YB-1 (D299), anti-HER2 (D8F12), anti-β-actin (13E5), anti-vinculin (E1E9V) and anti-Lamin B1 (D9V6H; Cell Signaling Technology, Beverly, MA, USA).
For whole-cell proteins studies, proteins were harvested by homogenizing cells in lysis buffer (50 mM Tris/HCl pH 8.5, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxycholate). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Equal amounts of samples were separated on 8% or 10% SDS-PAGE gels, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The blots were then probed with the appropriate HRP conjugated secondary antibodies and visualized using ECL (Thermo). The experiments were conducted at least three times. The results were quantified using Quantity One v4.62.
For subcellular localization studies, nuclear and cytoplasmic proteins were separated with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, Rockford, IL, USA) after whole-cell proteins extracted. Other steps of immunoblot were the same as whole-cell proteins studies.
For whole-cell proteins studies, proteins were harvested by homogenizing cells in lysis buffer (50 mM Tris/HCl pH 8.5, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxycholate). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Equal amounts of samples were separated on 8% or 10% SDS-PAGE gels, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The blots were then probed with the appropriate HRP conjugated secondary antibodies and visualized using ECL (Thermo). The experiments were conducted at least three times. The results were quantified using Quantity One v4.62.
For subcellular localization studies, nuclear and cytoplasmic proteins were separated with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, Rockford, IL, USA) after whole-cell proteins extracted. Other steps of immunoblot were the same as whole-cell proteins studies.