Igf 1rβ

Organotypic cultures were scraped from tissue culture plates and homogenized in RIPA lysis buffer that contained protease and phosphatase inhibitors (Thermo Scientific). Protein content of culture lysates was measured using Micro BCA protein assay kit (Thermo Scientific). Proteins were separated via electrophoresis in 12% Tris-Glycine Mini Gels (Life Technologies), transferred to a PVDF membrane, and stained with antibodies (1:10,000 rabbit antibodies to phospho-Akt (Ser473) (D9E), phospho-Akt (Thr308) (C31E5E), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E), phospho-S6 (Ser235/236) (D57.2.2E), phospho-S6 (Ser 240/244) (D68F8), phospho−IGF-1Rβ (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) (19H7, used at 1:500 concentration), total Akt (C67E7), p44/42 MAPK (Erk1/2) (137F5), S6 Ribosomal Protein (5G10), and IGF-1Rβ (D23H3, used at 1:1000 concentration), all from Cell Signaling Technology, and anti-rabbit horseradish peroxidase-conjugated secondary antibody from Jackson ImmunoResearch Laboratories). Bands were visualized using Pierce enhanced chemiluminescence (ECL) substrate on CL-XPosure X-ray films (Thermo Scientific), scanned, and quantified using densitometry via ImageJ software (NIH).

The anti-TM4SF4 antibody for the Western blot analysis was purchased from Sigma-Aldrich. Antibodies against Sox2, Phospho-AKT (Ser473), AKT, Phospho-NF-κB p65 (Ser536), NF-κB, Phospho-IGF1Rβ (Tyr1131), IGF1Rβ, integrin αV, CD44, and β-actin (Cell Signaling Technology, Danvers, MA, USA); Twist, c-Myc, Cyclin D1, β-catenin (Santa Cruz, Dallas, TX, USA), E-cadherin, and N-cadherin (BD Biosciences, San Jose, CA, USA); Vimentin (Thermo Fisher Scientific, Fremont, CA, USA); and Snail, Notch2, HSPA1L, ALDH1A1, ALDH1A3 (Abcam, Cambridge, UK), and Oct4 (Millipore, Billerica, MA, USA) were used. Protein concentrations were determined using a Lowry kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on 8% or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Hybond; Amersham Pharmacia). The blots were blocked for 1 h at room temperature with blocking buffer (10% nonfat milk in PBS containing 0.1% Tween 20). The membrane was incubated overnight in a cold chamber with specific antibodies. After being washed with TBS, blots were developed with a peroxidase conjugated secondary antibody, and proteins were visualized via enhanced chemiluminescence procedures (Amersham) following the manufacturer’s protocol.

Mouse tissues were crushed into a fine powder in liquid nitrogen and lysed in RIPA buffer supplemented with complete protease inhibitor (Roche, Switzerland). Protein concentration was determined using the BCA method (KeyGEN Bio TECH Nanjing, China). Western blot analysis was performed as previously described (23 (link)). Aliquots (20 μg) resolved by 8% SDS-PAGE gels electrophoresis, transferred to PVDF membrane (Pall, USA). After blocking with 5% non-fat milk in phosphate buffered saline/Tween-20, the membrane was incubated with antibodies specific to SHP-2 (1:1,000; #3397, Cell Signaling Technology, Boston, USA), CDC25A (1:1,000; #137353, Abcam, UK), mTOR (1:1,000; #2983, Cell Signaling Technology, Boston, USA), IGF1R-β (1:1,000; #9750, Cell Signaling Technology, Boston, USA). The antigen-antibody complexes were detected using an enhanced chemiluminescence (Advansta, Menlo Park, USA). The abundance assessed quantitatively using the Image J softwar.

Cav1 scaffolding domain peptide and control peptide [31 (link)] were purchased from Merck Millipore USA. The Src-family inhibitors SU6656 and PP2, PAR1-activating peptide (SFLLRN), PAR2-activating peptide (SLIGKV), geranyl–geranyl pyrophosphate (GGPP), and simvastatin were bought from Sigma-Aldrich Sweden AB. simvastatin was activated prior to the experiments by alkaline hydrolysis of the lactone ring according to the manufacturer’s protocol. Recombinant human FVIIa was a kind gift from Professor LC Petersen, NovoNordisk A/S, Denmark. The following antibodies were purchased: rabbit IGF-1Rβ, Cav1 rabbit, pSrc family Tyr416, Caspase-3, Cyclin-D1, β-actin, and pAbl Tyr245 from Cell Signaling Technology, USA; phosphotyrosine rabbit, HDAC-2 from Abcam, UK; Cav1 mouse from Santa Cruz Biotechnology, USA; Abl, activated β1 integrin (HUTS-4), and phosphotyrosine 4G10 Platinum from Merck Millipore; pCaveolin-1 Tyr14 from BD Transduction Lab, FITC-labeled activated β1 integrin from Bio-Rad, USA; FITC-labeled IgG from Dako Cytomation, USA; WB secondary antibodies IRDye 680CW (mouse and rabbit) and IRDye 800CW (mouse and rabbit) from LI-COR Biosciences, UK.