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2 protocols using anti egf receptor d38b1 xp rabbit mab

1

Western Blot Analysis of Immune Signaling Proteins

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The cells were washed with PBS and scraped using a cell scraper. The cells were transferred to 1.5 mL microtubes and centrifuged at 5000 g for 3 min. Cells were lysed in 100 μL of lysis buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 6% 2‐mercaptoethanol, 10% glycerol, 0.0125% bromothymol blue) and heated at 95°C for 5 min. Whole cell extracts were subjected to electrophoresis on 4%–20% Mini PROTEAN TGX gels (Bio‐Rad, 4561095) and then transferred to Trans‐Blot Turbo Transfer Pack membranes (0.2 μM PVDF, BIO‐RAD, 1704156). Membranes were blocked with 5% BSA in Tris‐Buffered Saline‐Tween, TBST 0.001% azide for 60 min and probed with primary anti‐STING (D2P2F) rabbit mAb (#13647, Cell Signaling Technology), anti‐cGAS (D1D3G) rabbit mAb (#15102, Cell Signaling Technology), anti‐EGF‐Receptor (D38B1) XP rabbit mAb (#4267), and anti‐β‐actin (13E5) rabbit mAb (#4970, Cell Signaling Technology). The membranes were then washed with TBST and incubated with a secondary donkey anti‐rabbit immunoglobulin antibody conjugated to horseradish peroxidase (HRP) (Abcam, ab205722) for 1 h. Proteins were detected using a chemiluminescence detection system (ECL Prime Western Blot Detection System; GE Healthcare, RPN2232) according to the manufacturer's instructions.
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2

Protein Extraction and Western Blot Analysis

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Proteins were extracted with a lysis buffer (TRIS-HCl 50 mM pH 8, NaCl 150 mM, Triton X-100 1%, NaF 100 mM, EDTA 1 mM, MgCl2 1 mM, Glycerol 10%) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and a phosphatase inhibitor cocktail (Roche) as previously described.40 (link) Equal amounts of total protein were subjected to SDS-PAGE and then electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in PBS with 0,1% Tween 20 and incubated overnight using the following antibodies: anti β-Actin A5441 (Sigma-Aldrich), anti-EGF Receptor (D38B1) XP Rabbit mAb (Cell Signaling, Danvers, MA, USA), anti-ErbB2 (D8F12) XP Rabbit mAb (Cell Signaling) anti-ZEB1 (Millipore, Life Science), anti-p63 Y4A3 (Sigma-Aldrich) or anti-p63 alpha (D2K8X) XP Rabbit mAb (Cell Signaling). After wash, membranes were hybridized with horseradish peroxidase-conjugated secondary antibodies (rabbit and mouse, Bio-Rad, Hercules, CA, USA). Detection was performed with Plus-ECL chemiluminescence kit (PerkinElmer, Inc., Waltham, MA, USA) or with SuperSignal West Dura extended duration substrate kit (Thermo Scientific, Waltham, MA, USA).
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