Erbb2

Polyclonal anti-TOM1L1 (1: 2,000) antibodies were as described in ref. 23 (link). The monoclonal anti-TOM1L1 antibody was from Covalab (Lyon, France). Antibodies against ERBB2 (1:1,000), p-CortactinY421 (1:500), p-AKTS473 (1:1,000), AKT (1:1,000), P44/P42 MAPK (1:1,000) and p-P44/P42 MAPK (1:1,000) were from Cell Signaling Technology (Danvers, USA). Antibody against Cortactin (1:500) was from Millipore (Billeria, USA) and antibodies against MT1-MMP (1:100 for if or 1:500 for immunoblotting) and TOLLIP (1:200) from Abcam (Cambridge, UK). Anti-tubulin (1:2,000), HA-tag (1:4,000) and p-Tyr 4G10 (1:50) antibodies were from N. Morin, C. Gauthier-Rouvière and P. Mangeat, respectively (CRBM, Montpellier, France). Anti-rabbit IgG-HRP (1:5,000) and anti-mouse IgG-HRP (1:5,000) were from GE Healthcare (Fairfield, USA). Anti-rabbit and anti-mouse IgG coupled to Alexa-Fluor 488, Alexa-Fluor 594 and Alexa-Fluor 405 (1:1,000) were from Life Technologies (Carlsbad, USA). Alexa-Fluor 594-Phalloidin (Life Technologies) was used to visualize F-actin. GFP-Nanotrap technology antibodies were from Chromotek (Planegg-Martinsried, Germany).

Cells were lysed in situ in hot Laemmli sample buffer [132 (link)]. Cell lysates were further incubated for 5 minutes at 95°C. Primary antibodies were obtained from Abcam (Cambridge, MA, USA): phospho-ErbB3 Y1222 (#ab133445) and Y1289 (#ab76469) or Cell Signaling Technology (Danvers, MA, USA): ErbB1 (#4267), ErbB2 (#2165), ErbB3 (#12708), phospho-ErbB1 Y845 (#6963), Y992 (#2235), Y1068 (#3777), Y1086 (#2220), Y1148 (#4404), phospho-ErbB2 Y1196 (#6942), phospho-ErbB3 Y1197 (#4561), Y1328 (#8017) and actin (#3700). Secondary antibodies were derived from LI-COR Biosciences (Lincoln, NE, USA): IRDye 800CW goat anti-mouse IgG (#926-32210) and IRDye 800CW goat anti-rabbit IgG (#926-32211). Immunoblots were analyzed and quantified using the Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Western blot analysis was performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in the RIPA buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were probed with the specific primary antibodies, followed by peroxidase-conjugated secondary antibodies. The bands were visualized by chemiluminescence (Denville Scientific). The following antibodies were used: antibodies to E-cadherin (1∶1000, BD Transduction Laboratories, 610182), vimentin (1∶2000, NeoMarkers, MS-129-P), Erbb2 (1∶500, Cell signaling Technology, 2242), HOXA1 (1∶1000, Santa Cruz Biotechnology, sc-17146), SMARCA5 (1∶500, sc-8760 from Santa Cruz Biotechnology and ab3749 from Abcam), SMARCD1 (1∶500, Abcam, ab86029), mTOR (1∶1000, Cell signaling Technology, 2972), BMPR2 (1∶1000, Cell signaling Technology, 69679), cyclin D1 (1∶1000, Cell signaling Technology, 2922), ALDH1A1 (1∶1000, Santa Cruz Biotechnology, sc-22589), HSP90 (1∶3000, BD Transduction Laboratories, 610419) and cyclophilin B (1∶2000, Thermo, PA1-027A).

Immunohistochemistry (IHC) and Immunofluorescence (IF) were performed as previously published^{57 (link)–59 (link)}. The following primary antibodies were used: BrdU (1:50, Becton Dickinson, 347583), PECAM (1:50, BD Pharmingen), MF20 (1:100, Developmental Studies Hybridoma Bank (DSHB)), NICD (1:50, Cell Signaling, 4147 S), ErbB2 (1:100, Cell Signaling, 2165 S), ESR (Abcam, pre-diluted), Endomucin (1:50, SCBT), p-Akt (1:50, Cell Signaling, 9271 S) and p-Erk (1:100, Cell Signaling, 4376 S).

Western blot and protein detection was performed as previously described [43 (link)]. The following primary antibodies were used: RB, phospho-RB^S807/S811, CDC25C, phospho-AKT^S473, phospho-cyclin E1^T62, phospho-ERK1/2^T202/Y204, ERBB2, phospho-ERBB2^Y1221/1222 and MYC (Cell Signaling, Danvers, MA); BRCA1, p53, cyclin E, CDK1, CDK2, ETV4, ETV5 and HRAS (Santa Cruz Biotechnology, Santa Cruz, CA); Actin (Abcam, Cambridge, MA); GAPDH (Fitzgerald, Acton, MA); β-Actin (Sigma-Aldrich); PAX8 (Epitomics, Burlingame, CA);

Ganetespib was purchased from Medkoo Biosciences, Inc. (Chapel Hill, NC). Lapatinib was purchased from LC Laboratories (Woburn, MA). Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA). Primary antibodies against CDK1, Cyclin D1, p27, caspase 8, caspase 9, HSP90, HSP70, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary mouse and rabbit antibodies were purchased from Thermo Scientific (Rockford, IL).

The expression of ErbB2, WT1, and programmed death ligand 1 (PDL1) in the GL261 cells before and after radiation was confirmed by western blotting. In general, the cells were exposed to 2, 4, or 6 Gy of radiation and cultured. The cells were harvested after the indicated time periods (0 and 24 h) for western blot analysis. The bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, USA) was used to measure protein concentration. Thereafter, SDS-PAGE was used to separate the proteins of interest, which were then transferred to a polyvinylidene difluoride (PVDF) membrane and soaked in a blocking solution [5% non-fat dry milk in TBST (tris-buffered saline, Tween 20)] for 1 h. The membrane was probed overnight with primary antibodies against WT1 (Abcam, Cambridge, UK), ErbB2 (Cell Signaling, Danvers, MA, USA), PDL1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-Actin (Santa Cruz Biotechnology) at 4°C, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse polyclonal IgG secondary antibodies (Ab Frontier, Seoul, Republic of Korea). Chemiluminescent detection was performed using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA). β-Actin was used as an internal control. The expression of WT1, ErbB2, and PDL1 was determined using Amersham Imager 600 (GE Healthcare, Marlborough, MA, USA).