Phospho egfr tyrosine 1068

CRP, total cholesterol, LDL, and HDL were measured by the UTMDACC Hospital Clinical Laboratory. Atorvastatin and its 2 hydroxylated metabolites (o-hydroxyatorvastatin and p-hydroxyatorvastatin) were measured in both serum and FNA samples using tandem mass spectrometers at the UTMDACC Pharmacology and Analytical CORE Laboratory as previously described [12 (link)].
Since certain HMG-CoAR genotypes can affect response to statins, we evaluated the rs12654264 gene for the A/A, A/W, or T/T genotypes and correlated with biomarker changes. The genotyping was carried at our institution’s DNA Analysis Core Facility.
Proliferation and apoptosis markers were immunostained in pre- and post-treatment FNA samples using unstained Thin-Prep slides. Staining for Ki-67 (ab66155, 1:100, Abcam), BCL-2 (ab692, 1:100, Abcam), and EGFR (CM063, 1:100, Biocare Medical). Phospho-EGFR (Tyrosine 1068) (2234, 1:50, Cell Signaling) was performed in the Pathology Immunohistochemistry Core Laboratory, according to the manufacturer’s instruction. Scoring of individual biomarker expression levels was performed by our 2 cytopathology collaborators (NS and YG), who recorded the percentage of positive-stained ductal cells.

The following antibodies were obtained from Cell Signaling Technology: phospho-EGFR tyrosine 1068 (#2234, 1:1000 dilution), EGFR (#4267, 1:1250 dilution), phospho-ERK threonine 202/tyrosine 204 (#9101, 1:2000 dilution), ERK (#9102, 1:2000 dilution), HRP-conjugated anti-mouse (#7076, 1:5000 dilution), and HRP-conjugated anti-rabbit (#7074, 1:5000 dilution). The actin antibody (#A2066, 1:5000 dilution) was purchased from Sigma-Aldrich. The EGFR antibody (#610017, 1:2000 dilution) was purchased from BD Pharmingen. For immunoblotting, cells were harvested, washed in PBS, and lysed in RIPA buffer (150 mM NaCl, 1% Triton-X-100, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM Tris·HCl, pH 8.0) with freshly added 40 mM NaF, 1 mM Na-orthovanadate, and protease inhibitor mini tablets (Thermo Scientific). Protein was quantified using protein assay reagent and a SmartSpec plus spectrophotometer (Bio-Rad) per the manufacturer's protocol. Lysates were subjected to SDS-PAGE followed by blotting with the indicated antibodies and detection by Western Lightning ECL reagent (Perkin Elmer).