Egfr d38b1

To analyze the protein expression levels, cell lysis was performed in buffer containing 0.5% Nonidet P40 (Roche, Mannheim, Germany, 11754599001), 100 mM Tris-HCl (pH 7.4), 300 mM NaCl, protease, and phosphatase inhibitors [20 (link),21 (link)]. Proteins were resolved by SDS-gel electrophoresis, and the blots were probed with the following antibodies: HRP-β-actin (AC-15) (ab49900, 1:20,000), cathepsin B (CTSB, ab58802, 1:1000), and LAMP1 (#24170, 1:1000) from Abcam, Cambridge, UK; CTSB (H-5) (sc-365558, 1:1000), CTSC (D-6) (sc-74590, 1:1000), CTSD (D-7) (sc-377299, 1:1000), LAMP1 (H4A3) (sc-20011, 1:1000) and LAMP2 (H4B4) (sc-18822, 1:1000) from Santa Cruz Biotechnology, Dallas, DX, USA; EEA1 (#2411, 1:1000), EGFR (D38B1) (#4267T, 1:1000), Rab5A (#2143, 1:1000), Rab7 (D95F2) (#9367, 1:1000), phosphor-AKT (ser473, #9271), phosphor-ERK1/2(Thr202/Tyr204, #9101), phosphor-mTOR (ser2448, #2971, 1:1000), phosphor-S6 ribosomal protein (D57.2.2E) (ser235/236, #4858, 1:2000), α-tubulin (DM1A) (TUBB, #3873S, 1:1000) from Cell Signaling Technology, Danvers, MA, USA; GAPDH (MAB374, 1:5000) from Merk-Millipore, Burlington, MA, USA; CTSL (CPL33/1) (C4618, 1:1000) and LC3 (L7543, 1:5000) from Sigma-Aldrich; and SQSTM1/p62 (#H00008878, 1:5000) from Abnova, Taibei, China.

Immunoblot analysis was as described previously [33 (link)]. Briefly, the cultured tumor cells were harvested and lysed in lysis buffer (50 mM Tris-HCI, pH 7.4, 50 mM NaCI, 1% Nonidet P-40, 2 mM EDTA, 10 mM NaF, 2 mM sodium orthovanadate and protease inhibitor cocktail). Whole cell lysate was electrophoresed on a 12% SDS-PAGE gel, transferred to nitrocellulose membrane (Bio-Rad Laboratories Inc., Hercules, CA) and immunoblotted with the a the phospho-EGFR (Tyr1068, D7A5; Cell Signaling), the EGFR (D38B1; Cell Signaling), or α-tubulin (YL1/2; Millipore, Temecula, CA). The intensity of the bands was quantified with ImageJ (Wayne Rasband, NIH, MD).
The cultured tumor cells were harvested and fixed. After washing with T-buffer once, the cell pellet was dissolved in a staining solution containing the PE-conjugated anti-CD326 (EpCAM) mAb 9C4 (1:25 dilution, BioLegend, San Diego, CA) or Alexa Fluor 647-conjugated anti-EGFR mAb D38B1 (Cell Signaling Technology). Samples were incubated overnight at 4°C in the dark. Unbound antibodies were removed via washing with 2 mL of T-buffer followed by centrifugation. Flow cytometry was performed using the On-chip Sort. Data analysis was performed using FlowJo software v7.6.5.