Ab52894

Bicinchoninic acid assay (BCA assay) relative protein quantification was used for normalization of sample loading. Proteins with 6 μg (EGFR, CD81, FGF-2, Flottin-1, Grp94, HSA), 8 μg (securin) or 10 μg (NLGN3) were loaded on per channel. Samples were separated using the assay of vertical slab polyacrylamide gel electrophoresis. After that, proteins were transferred onto polyvinylidene fluoride membranes (PVDF Invitrogen) using wet electrophoretic transfer. The membranes were blocked with 5% (w/v) skim milk and then incubated with primary antibody in 3 mL primary antibody dilution buffer. Next, the membranes were washed three times for 5 min each with 5 mL TBST followed by incubation with secondary IgG and HRP-linked antibody.
Western blot antibodies are listed below: Anti-EGFR, Abcam, ab52894, 1/5000. Anti-CD81, CST, 10037S, 1/1000. Anti-FGF2, Abcam, ab16828, 2 μg/ mL. Anti-Securin, Abcam, ab3350, 1/1000. Anti-HSA, Biorbyt, orb24991, 1/1000. Anti-Grp94, Sino Biological, 106461, 1/1000. Anti-Flotillin 1, Abcam, ab133497, 1/10000. Anti-CD81, CST, sc166029, 1/500. Anti-mouse IgG, HRP-linked Antibody, CST, #7076, 1/1000. Anti-rabbit IgG, HRP-linked Antibody, #7074, 1/1000.

The total protein of tissues and cells was extracted. After ice bathing and centrifugation, the sample was added to each lane for electrophoresis separation. The separated protein was transferred onto a nitrocellulose membrane. Then, the nitrocellulose membrane was blocked with 5% skim milk powder at 4°C overnight and incubated overnight with primary rabbit anti‐mouse polyclonal antibodies diluted at the ratio of 1:1000: NEK2 (ab115731), β‐catenin (ab16051), Nanog (ab21624), Oct‐4 (ab19857), CXCL16 (ab101404), EGFR (ab52894), TCF‐4 (ab217668) and Pygo2 (ab109001) and rabbit anti‐mouse monoclonal antibodies diluted at 1:1000: CD24 (ab64064), and CD44 (ab51037), all of which were purchased from Abcam Inc, Cambridge, UK. The membrane was washed 3 times with PBS at room temperature, 5 minutes for each, incubated with oscillation with the addition of the secondary antibody of horseradish peroxidase (HRP)‐labelled goat anti‐rabbit immunoglobulin (IgG) (1:10 000, ab6728, Boster Biological Technology Co. Ltd, Wuhan, China) at 37°C for 1 hour, washed with Tris‐buffered saline‐Tween (TBST) and visualized using HRP electroluminescence (ECL). Grey value analysis of the target bands was performed using ImageJ software, and the experiment was repeated three times independently.

The protein concentration was determined by cellular protein extraction, ensuring that the amount of protein was not less than 1 mg. A small amount of lysate was taken as input for Western blot analysis, and 1 μg of the corresponding target protein antibody anti-EGFR (1:20, ab52894, Abcam), anti-PTEN (1:500, 9559, Cell Signaling Technology) and 80 μL of ProteinA/proteinG magnetic beads were incubated overnight at 4 °C with slow shaking. After the immunoprecipitation, the magnetic beads were centrifuged to the bottom of the tube, the supernatant was aspirated and discarded. Cells were eluted with 3 × SDS loading buffer. The lysis buffer contained 50 mM heparin (pH 7.4), 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% glycerol, protease and phosphatase inhibitors. After addition of lysis buffer, lysates were incubated on ice for 30 min without sonication and then removed by centrifugation. Western blot assay was applied for further detection.

Paraffin sections of clinical tissue specimens were taken, dewaxed to water, dehydrated in alcohol gradient, and repaired in water bath in antigen repair solution. Normal goat serum blocking solution (C-0005, Haoran Biotechnology, Shanghai, China) was added to the sections at room temperature for 20 min, followed by addition of primary antibodies of rabbit anti-human EGFR (1:100, ab52894, Abcam) and p-EGFR (1:100, ab5652, Abcam) overnight at 4 °C. Then sections were added with goat anti-rabbit immunoglobulin G (IgG) (ab6785, 1:1000, Abcam) secondary antibody. The tissues were placed at 37 °C for 20 min, and dripped with horseradish peroxidase-labeled streptavidin protein working solution (0343-10000U, Emo Biotech, Beijing, China) which was placed at 37 °C for 20 min. Sections were counterstained with DAB (ST033, Weijia Technology, Shanghai, China) for 1 min and returned blue with 1% ammonia. Sections were observed under a microscope and filmed with 5 high magnification fields randomly selected for each section and 100 cells were selected in each field.

The following antibodies were used:
Rabbit monoclonal anti-EGFR (ab52894, Abcam, Cambridge, UK); mouse monoclonal anti-EGFR (sc-120, Santa Cruz Biotechnology, USA); mouse monoclonal anti-EGFRvIII (L8A4, Absolute Antibodies, UK); rabbit polyclonal anti-EGFR (ab5652, Abcam, UK); mouse monoclonal anti-HO-1 (ab13248, Abcam, UK); mouse monoclonal anti-β-actin (AC-15, Sigma-Aldrich, USA); IRDye^® 800CW goat anti-mouse IgG (827-08364, LI-COR Biotechnology, Germany); IRDye^® 680RD goat anti-rabbit IgG (926-68071, LI-COR Biotechnology, Germany).

Fibroblasts lysates were prepared in RIPA buffer (R0278 Sigma) with complete protease inhibitor cocktail (Roche). Protein concentration in each sample was determined with Bradford protein assay and equal quantity of protein was loaded in Mini-PROTEAN 4–20% Tris-Glycine SDS-PAGE (Biorad) and transferred on nitrocellulose membranes (Santa Cruz). After incubation in TBS/non-fat milk 5% for 1 h, membranes were incubated in anti-EGF-R (ab52894, Abcam, 1/1000) or anti-GAPDH (CB1001, Millipore, 1/6000) diluted in TBS/Tween 0.1%/BSA 5% overnight at 4 °C. Membranes incubated in horseradish peroxidase conjugated secondary antibodies anti-rabbit (1:5000, 31,460, Thermofisher Scientific) or anti-mouse (1:5000, 31,430, Thermofisher Scientific) diluted in TBS/Tween 0.1% for 1 h, incubated in ECL substrate (34,580, Thermofisher Scientific) and developed on ECL films (Amersham Hyperfilm, GE). EGF-R level was normalized to GAPDH level in each condition.

The total protein was extracted from tissues or cells with radioimmunoprecipitation assay lysis buffer (R0010, Solarbio), with the concentration determined by BCA Kit (20201ES76, YEASEN Biotech Co., Ltd., Shanghai). After separation by polyacrylamide gel electrophoresis, the protein was transferred to the PVDF membrane by wet transfer method. The membrane was sealed with 5% BSA at room temperature for 1 h, probed with the primary antibodies to CMTM7 (#PA5-103744, 1:1000, Thermo Fisher), EGFR (ab52894, 1:1000, Abcam), p-EGFR (ab40815, 1:1000, Abcam), AKT (ab8805, 1:500, Abcam), p-AKT (ab8933, 1:500, Abcam), VEGF (ab32152, 1:1000), CD63 (ab134045, 1:1000, Abcam), Hsp70 (ab181606, 1:1000, Abcam), TSG101 (ab125011, 1:2000, Abcam), and Calnexin (ab133615, 1:1000, Abcam) at 4 °C overnight. The next day, the membrane was re-probed with HRP labeled goat anti-rabbit IgG (ab205718, 1:10,000, Abcam) for 1 h at room temperature, developed by VILBER FUSION FX5 (VILBER LOURMAT, France). Image J 1.48u software (National Institutes of Health) was used for protein quantitative analysis, and the gray value of each protein was compared with the gray value of internal reference GAPDH.