Anti lamin b1 d9v6h

Cells modified with SB transposons were seeded in 12‐well plates. 24 h after transfection protein expression was induced by doxycycline (1 μg/mL). Next day cells were lysed in SDS Loading Buffer (0.35 M Tris·HCl, 35% (v/v) glycerol, 10% (w/v) SDS, 3.6 M β‐mercaptoethanol, 0.12 g/mL bromophenol blue) and denatured in 95°C for 7 min. Protein extracts were analyzed by Western blot. Following antibodies were used: mouse monoclonal anti‐FLAG (F3165, Merck), anti‐HA‐Tag (C29F4, Cell Signaling Technology), anti‐Lamin B1 (D9V6H, Cell Signaling Technology), anti‐GAPDH (D16H11, Cell Signaling Technology), anti‐IκBα (L35A5, Cell Signaling Technology), anti‐β‐actin (8H10D10, Cell Signaling Technology), anti‐GFP (A‐11122, Invitrogen) anti‐mouseHRP (#7076), and anti‐rabbit HRP (#7074) (Cell Signaling). Luminescence was detected using the Clarity Western ECL Substrate (Bio‐Rad) and recorded using the Fusion‐Fx documentation system (Vilber Lourmat).

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.