Extra page one precast gel 5 20

Preparation of total cell lysate and western blot analysis were carried out as previously described [31 , 36 (link), 37 (link)] with the following modifications: The total proteins were electrophoresed on Extra PAGE One Precast Gel 5–20% (Nacalai Tesque, Kyoto, Japan, 13064‐64); the molecular weight marker used was a 3‐Color prestained XL‐ladder (APRO Science, Tokushima, Japan, SP‐2140); and the membranes were blocked in Blocking One (Nacalai Tesque, 03953‐95) or ECL Prime Blocking reagent (GE Healthcare Bio‐Sciences. Corp., Piscataway, NJ, USA, RPN418) for 30 min at room temperature. The following antibodies were used: goat anti‐XRCC4 polyclonal antibody (C‐20; Santa Cruz Biotechnology, Santa Cruz, TX, USA, sc‐8285), anti‐C9orf142 (PAXX) polyclonal antibody (Abcam, Cambridge, UK, ab126353), rabbit anti‐GFP polyclonal antibody (FL; Santa Cruz Biotechnology, sc‐8334), and mouse anti‐β‐actin monoclonal antibody (AC‐15; Sigma‐Aldrich, St. Louis, MO, USA, A5441). In accordance with the manufacturer's instructions, protein bands were detected using a Select Western Blotting Detection System (GE Healthcare Bio‐Sciences. Corp., RPN2235) or Chemi‐Lumi One Ultra (Nacalai Tesque, 11644‐40), and visualized using the ChemiDoc XRS System (Bio‐Rad, Hercules, CA, USA).

Cell pellet and tumour fragments were lysed in lysis buffer (0.1 M Tris–HCl pH 7.5, 10% glycerol and 1% sodium dodecyl sulfate), boiled for 5 minutes and centrifuged for 10 minutes (15,000 rpm). All protein concentrations were determined by BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). Each cell lysate (10 μg) was electrophoresed on Extra PAGE One Precast Gel 5–20% (Nacalai Tesque) and transferred onto PVDF membrane (Millipore Corp., Billerica, MA). After blocking with 5% skim milk (Megumilk, Tokyo, Japan) or 5% bovine serum albumin (Nacalai Tesque), the membrane was treated with primary antibodies to podoplanin (FL-162; Santa Cruz Biotechnology, CA, USA), EGFR (phospho Y1068) (Abcam, Cambridge, MA, USA), GAPDH (Millipore), β-actin (clone, AC-15, Sigma-Aldrich), EGFR and pErk (Cell Signaling Technology, Boston, MA, USA). After treating with an ECL Prime Western Blotting Detection reagent (GE Healthcare), we used LAS-3000 mini (Fujifilm, Tokyo, Japan) to detect chemiluminescence signals.