Anti egfr

Whole cell lysates and EVs (4 × 10⁸ EVs) were derived from PalmReNL-HEK293FT cells and mixed with 4× sample buffer (Bio-Rad (Hercules, CA, USA)) with β-mercaptoethanol for detecting Alix, CD9, TSG101, and tdTomato proteins. Whole cell lysates derived from MDA-MB-231 cells were mixed with 2× sample buffer without β-mercaptoethanol (non-reducing conditions) for detecting PDL1, uPAR, EGFR, and GAPDH. Proteins were separated on a 4–20% Mini-PROTEAN TGX gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Millipore, IPFL00010). After blocking with 5% non-fat dry milk at RT for 1 h, membranes were probed with primary antibodies overnight at 4 °C at dilutions recommended by the suppliers as follows: anti-Alix (Proteintech (Rosemont, IL, USA), 12422-1-AP), anti-TSG101 (Proteintech, 14497-1-AP), anti-CD9 (Proteintech, 60232-1-lg), anti-RFP (Rockland Immunochemicals (Pottstown, PA, USA), 600-401-379), anti-PDL1 (Proteintech, 66248-1-lg), anti-uPAR (Proteintech, 10286-1-AP), anti-EGFR (Proteintech, 18986-1-AP), or anti-GAPDH (Santa Cruz (Santa Cruz, CA, USA), G-9), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. The membranes were visualized with ECL select Western Blotting Detection Reagent (GE Healthcare (Chicago, IL, USA), RPN2235) on ChemiDoc MP Imaging System (Bio-Rad).

Total protein from AGS and HGC27 cells was extracted using commercial kit, and the protein concentration was determined with BCA method. The samples were subjected to SDS–PAGE, and were transferred to the PDVF membrane that were placed in 5% BSA solution (ThermoFisher Scientific, MA, USA). The primary antibodies were incubated at four degrees overnight. The primary antibodies utilized in the experiments included anti-BAX (Proteintech, 50599-2-Ig, 1:1000), anti-β-action (Proteintech, 66009-1-Ig, 1:5000), anti-Bcl-2 (Proteintech, 68103-1-Ig, 1:1000), anti-PARP (Cell Signaling Technology, #9532, 1:1000), anti-Cleaved PARP (Cell Signaling Technology, #5625, 1:1000), anti-Caspase-3 (Cell Signaling Technology, #14220, 1:1000), anti-Cleaved Caspase-3 (Cell Signaling Technology, #9664, 1:1000), anti-PI3K (Affinity, AF6241, 1:1000), anti-p-PI3K (Affinity, AF3242, 1:1000), anti-AKT (Cell Signaling Technology, #4691, 1:1000), anti-p-AKT (Cell Signaling Technology, #4060, 1:1000), anti-EGFR (Proteintech, 66455-1-Ig, 1:1000), Anti-Cyt C (Proteintech, 10993-1-AP, 1:1000). The secondary antibodies were added and incubated at room temperature for 1 h. Finally, the bands were visualized using the IMAGE J software, with β-action serving as the internal standard.

Whole cell proteins were harvested and the protein concentrations were measured using a BCA kit (Pierce; Thermo Fisher Scientific, Inc). And equal protein (30 μg) was separated with 10% SDS‐PAGE and transferred onto nitrocellulose membranes (EMD Millipore), subsequently, the membranes were then blocked in 5% non‐fat dry milk for 2 hours at room temperature. The membrane was incubated with primary antibodies at 4°C overnight, and the membrane was washed with TBST and incubated with anti‐HPR‐conjugated Affinipure goat anti‐rabbit/mouse lgG(H+L) (1:5000, Proteintech Group, Inc). The signals were detected using an ECL Western blotting kit. The primary antibodies used included: Anti‐B3GNT5 (1:500), anti‐GAPDH (1:3000), anti‐cyclinD1 (1:500), anti‐cyclinB1 (1:500 proteintech), anti‐EGFR (1:1000) (Proteintech Group, Inc) anti‐ERK (1:1000), anti‐p‐ERK (1:500) (Cell Signaling Technology, Inc); AKT (1:1000, Beyotime Institute of Biotechnology) and p‐AKT(Ser473) (1:500, Beyotime Institute of Biotechnology).

Immunofluorescence was performed as previously described^{36 (link)}. Images were taken using ZEN2010 on Zeiss LSM-780 confocal microscopy. Co-localization analysis was performed using ZEN2.3 software. The following primary antibodies were used: anti-Flag (1:200, Sigma-Aldrich, F3165), anti-HA (1:200, Roche, 11867423001), anti-GM130 (1:200, Cell Signaling Technology, #12480 s), anti-GM130 (1:200, BD, 610822), and anti-EGFR (1:100, Proteintech, 18986-1-AP). The secondary antibodies were: Donkey anti-Mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific, A21202), Goat anti-mouse IgG Alexa Fluor 647 (Thermo Fisher Scientific, A21236), Goat anti-Rat IgG Alexa Fluor 594 (Thermo Fisher Scientific, A11007), Donkey anti-Rabbit IgG Alexa Fluor 647 (Thermo Fisher Scientific, A31573), and Goat anti-Rabbit IgG Alexa Fluor Plus 555 (Thermo Fisher Scientific, A21429). FITC or Rhodamine-labeled phalloidin (1:80, ABclonal Technology, RM02836, RM02835) were used to stain F-actin to indicate localization of plasma membrane. For the Proximity ligation assay, Rabbit anti-HA antibody (1:200, Cell Signaling Technology, #3724) and mouse anti-Flag antibody (1:200, Sigma-Aldrich, F3165) were used as primary antibodies. Duolink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich) was used following the manufacturer’s instructions.

Western blotting was performed as previously described [29 (link)]. Protein samples were separated by SDS–PAGE electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the PVDF membranes with proteins were incubated with 5% nonfat milk, specific primary antibodies at 4 °C overnight, and secondary antibodies (1:5000, BA1050, BA1054, Boster, Wuhan, China) in sequence and detected using ECL reagent. The primary antibodies were as follows: anti-Annexin A1 (1:1000, 32,934, Cell Signaling Technology), anti-E-cadherin (1:1000, ab231303; Abcam, Cambridge, U.K.), anti-vimentin (1:1000, ab20346; Abcam), anti-MMP9 (1:1000, ab76003; Abcam), anti-EGFR (1:1000, 66,455–1-Ig; Proteintech), anti-phospho-EGFR (1:1000, 3777, Cell Signaling Technology), anti-AKT(1:1000, 4685, Cell Signaling Technology), anti-phospho-AKT (1:1000, 4060, Cell Signaling Technology), anti-ERK (1:1000, 4659, Cell Signaling Technology), anti-phospho-ERK (1:1000, 4370, Cell Signaling Technology), anti-STAT3, anti-phospho-STAT3, anti-GAPDH (1:10,000, 60004–1-Ig; Proteintech), and anti-alpha tubulin(1:10,000, 66031-1-Ig; Proteintech) antibodies.

Anti-p-EGFR 1/2 (Y1068) (1:1,000, #3777s), anti-p-Erk (T202/Y204) (1:1,000, #4370s), anti-Erk (1:1,000, #4695s), anti-p-stat3 (Y727) (1:1,000, #9136), anti-stat3 (1:1,000, #4904), anti-p-Akt (S473) (1:500, #4060s), anti-PARP (1:1,000, #9532s), and anti-Cleaved PARP (1:1,000, #5625s) were purchased from Cell Signaling Technology. Anti-EGFR (1:1,000, 18986-1-AP), anti-Akt (1:1,000, 10176-2-AP), and anti-tubulin (1:1,000, 66031-1-Ig) were purchased from Proteintech. Anti-Flag (1:1,000, Mouse IgG, F1804), polybrene, and cycloheximide (CHX) were purchased from Sigma-Aldrich. Osimertinib and gefitinib were purchased from MedChemExpress (MCE).

Human hepatocellular carcinomas cells Huh-7 and HepG2 were cultured in 6-well plates and cultured overnight. Then we treated with dihydrotanshinone I (0, 2.5, and 5.0 µM) for 48 h. Cells were lysed with lysis buffer and the protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Samples were then subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (0.25 µm, Millipore, Burlington, MA, USA). The membrane was blocked in fresh 5% nonfat milk for 1.5 h at room temperature. Samples were followed incubated with anti-p-EGFR (CST, D38B1, 1:1000), anti-EGFR (CST, D7A5, 1:1000), anti-p-STAT3 (CST, D3A7, 1:1000), anti-STAT3 (CST, 124H6, 1:1000), anti-p-AKT (CST, 11E7, 1:1000), anti-BAX (CST, 41162S, 1:1000), anti-Bcl2 (CST, 15071S, 1:1000) and anti-AKT (Proteintech, 66444, 1:1000) primary antibodies at 4 °C overnight. After washing with PBST (3 × 10 min), samples were incubated with peroxidase-conjugated secondary antibodies (7076S and 7074S) for 1.5 h. Finally, proteins were detected using an enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA).