Total pi3k

Immunohistochemical staining of human GBM tissue specimens and mouse xenograft tumor samples were performed with antibodies against EGFR (Abcam), total PI3K (Cell Signaling Technology), p-PI3K (Cell Signaling Technology), total AKT (Cell Signaling Technology), p-AKT (Cell Signaling Technology), total-mTOR (Abcam), p-mTOR (Abcam), total-p70S6K (Abcam), p-p70S6K (Abcam), cyclin E (Cell Signaling Technology), CDK4 (Cell Signaling Technology), and Ki67 (Abcam) as described previously [31 (link)].

Nasal fibroblasts were treated with CSE for 72 h. A total of 5 × 10⁵ fibroblasts were lysed in PRO-PREP^TM protein extraction solution (iNtRON Biotechnology) and stored overnight at −20 °C. Cell debris was removed from the lysates by centrifugation at 13,000× g for 30 min at 4 °C. Total protein concentration was determined using the Bradford assay (Bio-Rad). An equal quantity of protein from samples (30 µg) were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to 0.45 μm polyvinyl difluoride membranes, (Millipore Inc., Billerica, MA, USA), and analyzed separately. Membranes were blocked with 5% skim milk at room temperature for 60 min, rinsed three times with Tris-buffered saline containing Tween-20, and treated with the following primary antibodies: polyclonal p-PI3K (1:1000, #4228, Cell Signaling Technology, Danvers, MA, USA), total-PI3K (1:1000, #4292, Cell Signaling Technology), p-Akt (1:1000, #9271, Cell Signaling Technology), total-Akt (1:1000, #9272, Cell Signaling Technology), p-p65 (1:1000, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), total-p65 (1:1000, sc-8008, Santa Cruz Biotechnology Inc.) and β-actin (1:10000, sc-47778, Santa Cruz Biotechnology Inc.). Bands were visualized using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence system (Pierce, Rockford, IL, USA).

Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), Antibiotic-Antimycotic (#15240096), GlutaMAX™ l-alanyl-l-glutamine dipeptide (#35050061), phosphate-buffered saline (PBS; pH 7.4), and 0.25% trypsin-EDTA were obtained from Gibco (Gaithersburg, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Triton X-100, Ribonuclease A (RNase A), crystal violet, paraformaldehyde, wortmannin, Hoechst 33342 and propidium iodide (PI) were purchased from Sigma-Aldrich Chemical, Co. (St. Louis, MO, USA). A radioimmunoprecipitation assay (RIPA) lysis buffer and Immobilon Western Chemiluminescent HRP Substrate were purchased from Merck (Darmstadt, Germany), and the protease inhibitor cocktail was purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). A bicinchoninic acid (BCA) protein assay kit was purchased from Thermo scientific (Rockford, IL, USA). The primary antibodies against p-PI3K (Tyr485/Tyr199; #4228), total PI3K (#4292), p-Akt (Ser473; #4060), total Akt (#9272), Met (#8198), p-GSK-3β (Ser9; #9332), total GSK-3β (#9832), β-catenin (#8480), c-Myc (#5605), β-actin (#4970), and the secondary antibody anti-rabbit IgG (#7074) or anti-mouse IgG (#7076) were acquired from Cell Signaling Technology (Danvers, MA, USA).

The protein expression levels were analyzed by western blotting as described in our previous studies (Park et al., 2020 (link)). Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary antibodies against MMP-2 (Cell signaling #4022), MMP-9 (Cell Signaling #13667), phospho-PI3K (Cell Signaling #4228), total PI3K (Cell Signaling #4292), phospho-Akt (Cell Signaling #4060), total Akt (Cell Signaling #4491) and β-actin (Abcam, MA, United States, ab189073) overnight at 4°C and then with polyclonal HRP-conjugated goat anti-mouse IgG (BD Pharmingen, 554002) or goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, United States, 554021) secondary antibodies at room temperature for 60 min. The antigen-antibody complexes were detected using Western blot ECL reagents (GE Healthcare, Bucks, United Kingdom).

Agrin (D2) and rabbit (H-300), Lrp4, Cdc42, N-WASP (western blot (WB) dilution—1:100) and anti-GFP (WB dilution—1:1,000) antibodies were obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA), Antibodies against Na+/K+ ATPase, Rab5, integrin β1, cleaved caspase-3, total FAK, cortactin, pSrc, total Src, pPI3-K, total PI3-K, pAkt, total Akt, pERK1/2 and total ERK1/2 were obtained from Cell Signaling Technology (dilution for WB—1:1,000). Antibodies against pFAK pY397, caveolin-1, Ki67, E-cadherin, N-cadherin and Vimentin were purchased from BD Biosciences, San Jose, CA (WB dilution—1:500). PF-562271, glypican-3, flotillin-1, Arp2/3 subunit 1B, pFAK (pY397), Snail-1 and MuSK antibodies were obtained from Abcam (dilution for WB—1:500). Phospho-tyrosine monoclonal antibody 4G10, Agrin monoclonal antibody MAb5204, p34Arc, GFP and EGFP antibodies were obtained from Millipore, Billerica, MA (WB dilutions—1:1,000). Agrin polyclonal rabbit antibody, OKT-9 hybridoma cells producing anti-CD-71 antibody and fibronectin antibody were obtained from Sigma, St Louis, MO. Recombinant rat Agrin protein residues was obtained from R&D systems and My Biosource, Inc, San Diego, CA. Recombinant CTxB, Alexa 488 and 555 conjugated secondary antibodies were from purchased Molecular Probes, Invitrogen, Carlsbad, CA.