Ab5644

Cells lysis was performed with total protein extraction buffer (NCM Biotech, Suzhou, China), and tissues were homogenized in T-PER tissue protein extraction reagent (Thermo Fisher Scientific), to which a combination of protease and phosphatase inhibitors was added (NCM Biotech, Suzhou, China). The protein concentration was measured by a BCA Protein Assay Kit (Thermo Fisher Scientific). Cell or tissue lysates (approximately 20 μg of protein) were subjected to SDS-PAGE (8–10%) and transferred to nitrocellulose (NC) membranes. After blocking with 5% fat-free milk, the NC membranes were incubated with different primary antibodies at 4 °C overnight. Antibodies against the following proteins were used: GPAA1 (1:1000, Bioss, bs-13496R), caveolin-1 (1:1000, Abcam, ab2910), AP2B1 (1:1000, Proteintech, 15,690–1-AP), β-actin (1:10000, Abcam, ab5644), EGFR (1:1000, Abcam, ab52894), p-EGFR (Y1068) (1:1000, Abcam, ab40815); ERBB2 (1:1000, Abcam, ab16901); p-ERBB2 (Y877) (1:1000, Abcam, ab47262), p-AKT (S473) (1:1000, CST, #4060), and AKT (1:1000, CST, #4685). After three washes in TBST (pH 7.4), membranes were incubated with species-specific secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 h. Ultimately, protein bands were visualized by an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Total cellular protein was isolated with RIPA buffer (Beyotime Institute of Biotechnology), and a BCA assay kit (Beyotime Institute of Biotechnology) was then utilized to quantify protein levels in each sample. Equivalent protein amounts were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). These blots were then blocked for 2 h with 5% non-fat milk, followed by 4 °C incubation overnight with appropriate primary antibodies. After being washed, blots were then incubated with an HRP-conjugated antibody (1:5,000; ab205718; Abcam), after which protein levels were visualized using an Immobilon Western Chemiluminescent HRP Substrate kit (Millipore). Primary antibodies were: rabbit polyclonal anti-EGFR (1:1000, ab5644, Abcam), anti-PTEN (1:1000, ab32199, Abcam), anti-Akt (#9272, Cell Signaling Technology, USA), anti-phospho (Ser473)-Akt (1:2000, #4060, Cell Signaling Technology), anti-MMP7 (1:1000, ab5706, Abcam), anti-MMP9 (1:1000, ab38898, Abcam), anti-E-Cadherin (1:1000, ab1416, Abcam), anti-N-Cadherin (1:1000, ab18203, Abcam), anti-Vimentin (1:1000, ab92547, Abcam).