Anti p egfr y1068

Biopsy fragments from duodenum obtained from GCD–CeD and organoids, were processed [10 (link),28 (link)] After removing matrigel by Cell Recovery Solution (cat. 354270, Corning Milan Italy), organoids were homogenized in 50 µL tissue homogenization buffer [28 (link)]. The cell lysates (10 μg/mL) were analysedby SDS-PAGE and transferred onto nitrocellulose membranes by Trans Blot Turbo (cat.1704158, BioRad, Milan, Italy). The membranes were blocked with 5% non-fat dry milk and probed with anti-PTPRk (cat. ab222249, Abcam, Microtech, Naples, Italy) and rabbit anti-pY-ERK1/2, (cat.20869, Elabscience, Microtech, Naples, Italy), anti-P-EGFR (Y1068) (cat.3777,Cell Signalling, Euroclone Milan, Italy) and anti-EGFR (cat.2232, Cell Signalling, Euroclone Milan, Italy), mouse anti-GAPDH, (cat. G8795, Sigma-Aldrich, Milan, Italy) and with rabbit anti-ERK1/2 (cat.31374, Elabscience, Microtech, Naples, Italy) were used as loading controls for biopsies and organoids, respectively. Then, two 10 min exposure using ECL (cat. RPN2209, GE Healthcare, Amersham, Buckinghamshire, UK) allowed to visualise the bands of interest. The bands intensity was evaluated by integrating all the pixels of the band after subtraction of the background to calculate the average of the pixels surrounding the band.

The anti-CLEC11A monoclonal antibody (Cat# MAB1904) was purchased from R&D System (Minneapolis, MN, USA). The anti-FAK (Cat# sc-1688), anti-integrins α2 (Cat# sc-6586), α5 (Cat# sc-10729), and β1 (Cat# sc-6622), p130-Cas (Cat# sc-20029), and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). The anti-active integrin β1 antibody (Cat# 550531) was obtained from BD Pharmingen (San Diego, CA, USA). The anti-EGFR (Cat#2232), anti-pEGFR (Y1068, Cat# 2234), anti-pFAK (Y576/577, Cat# 3281), and anti-pp130-Cas (Y165, Cat# 4015) were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-pFAK (Y397; Cat# ab81298) antibody was purchased from Abcam (Cambridge, UK). The anti-actin (Cat# A5441) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-TGFBI (Cat# GTX100744) and anti-tubulin (Cat# GTX62882) antibodies were obtained from GeneTex (Hsinchu, Taiwan). G418 and Zeocin were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Invitrogen (Carlsbad, CA, USA), respectively. VEGF-A 165 (Cat# 292-VE) and basic FGF (Cat# 234-FSE) were purchased from R&D System (Minneapolis, MN, USA).

Anti-pEGFR (Y1068) (#2234, 1:5000), EGFR (#4267, 1:5000), pYAP (S127) (#4911, 1:2000), YAP (#14074, 1:1000), YAP/TAZ (#8418, 1:1000), pLATS1 (T1079) (#8654, 1:1000), LATS1 (#3477, 1:1000), LATS2 (#5888, 1:1000), pMST1/2 (T183/180) (#3681, 1:1000), MST1 (#3682, 1:1000), pMOB1 (T35) (#8699, 1:1000), MOB1 (#13730, 1:1000), TEAD1 (#12292, 1:1000), HA-tag (#3724, 1:10000), Myc-tag (#2276, 1:5000), FLAG-tag (#2368, 1:5000), GST-tag (#2624, 1:10000), pTyrosine (P-Y-100) (#9411, 1:2000), CTGF (#86641, 1:1000), CYR61 (#14479, 1:1000), pERK1/2 (T202/Y204) (#4370, 1:10000), ERK1/2 (#4696, 1:10000), pAKT (S473) (#4060, 1:5000), AKT (#9272, 1:5000), pS6 (S235/236) (#4858, 1:10000), S6 (#2217, 1:10000), PARP (#9542, 1:1000), GRB2 (#36344, 1:1000), β-actin (#4967, 1:5000) were purchased from Cell Signaling Technology (MA).
EGF (#E9644) was purchased from Sigma-Aldrich Inc (MO). BYL719 (#16986) was purchased from Cayman Chemical (MI).