Irdye 800cw goat anti rabbit 926 32 211

The antigen peptides for all LRIG1 antibodies used are indicated in Supplementary Fig. 1B. For immunohistochemistry: Anti-LRIG1 antibody (Atlas Antibodies AB, Bromma, Sweden, catalog no. HPA011846), 4.0 mg/ml. The 1A8 anti-LRIG1 antibody is described in the Supplementary Materials. For western blots: EGFR #2232, 1:1000; Phospho-p44/42 MAPK #4370, 1:1000; Phospho-AKT #4060, 1:1000 (Cell Signaling Technology Inc, Danvers, USA); Anti-Actin #ACTN05/C4, 1:3000 (Abcam, Cambridge, UK). Anti-LRIG1 Vina [49 (link)], 1:1000 (Agrisera AB, Vännäs, Sweden). Secondary antibodies: IRDye^® 680RD Donkey anti-Mouse #926-68072, 1:15000; IRDye^® 800CW Goat anti-Rabbit #926-32211, 1:15000 (LI-COR, Lincoln, NE, USA).

Cells were collected and lysed with NP-40 buffer (Beyotime). The protein lysis was boiled for 20 min at 100 C in SDS-PAGE loading buffer. Equal amounts of cell lysate were 10% polyacrylamide-SDS gels. Proteins were transferred onto a PVDF membrane, blocked with 5% skim milk, and probed with anti-ORF1p antibody (diluted 1:1000) and anti-β-actin antibody (diluted 1:5,000) at 4°C overnight. After four wash steps with PBS plus 0.1% Tween 20 (PBST), the membrane was incubated with 1:5,000 dilution of HRP-conjugated goat-anti-mouse, HRP-conjugated goat-anti-rabbit, IRDye^® 800CW Goat anti-Rabbit 926–32,211 (1:10,000, LI-COR) and IRDye^® 700CW Goat anti-Mouse 926–32,210 (1:10,000, LI-COR) secondary antibodies for 1 h at room temperature. After four wash steps with PBS plus 0.1% Tween 20 (PBST), signals were detected using Western Lightning chemiluminescence reagent or analyzed with the Odyssey infrared imaging system and the software program as specified in the Odyssey software manual.

Red protein G affinity beads, RIPA buffer, and protease inhibitor cocktail were purchased from Sigma-Aldrich Corporation (St. Louis, MO). The HSP90 inhibitors, AUY-922 and AT13387, were obtained from Selleck Chemicals (Houston, TX). The BCA Protein Assay Kit was purchased from Pierce Co. (Rockford, IL), and Western blot membranes were purchased from GE Healthcare (Chicago, IL). All antibodies used in Western blots and immunoprecipitation have published immunospecificity data available online. Rabbit anti-AKT, anti-phospho AKT, anti-IKBα, anti-phospho IKBα, anti-STAT3, anti-phospho STAT3, anti-VE-cadherin, anti-occludin, anti-cofilin, and anti-phospho cofilin were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti-β-actin and anti-p53 (P8999) were purchased from Sigma-Aldrich Corporation, and secondary IRDye 800CW goat anti-rabbit (926-32211) and IRDye 680RD goat anti-mouse (926-68070) were purchased from LI-COR Biosciences (Lincoln, NE). For SDS-PAGE, Protogel (30% acrylamide mix) and TEMED were purchased from National Diagnostics (Atlanta, GA), Tris-HCl buffer was purchased from Teknova (Hollister, CA), 10% SDS and ammonium persulfate were purchased from Thermo Fisher Scientific, and protein dual-color standards and tricine sample buffer were purchased from Bio-Rad Laboratories.

Cell cultures were seeded into 6 well plates and trypsinized after 7 days of differentiation. Then, cell pellets were solubilized in lysis/loading buffer and denatured at 95 °C for 5 min. Samples were loaded onto a NuPAGE® Novex® 3–8% Tris–Acetate Gel3–8% (Thermo Fisher Scientific) and run in Novex Tris–Acetate SDS Running Buffer (Thermo Fisher Scientific) for 60 min at 70 V + 120 min at 150 V at 4 °C. Protein wet transfer was performed overnight at 4 °C using an Immobilon®-FL PVDF membrane (Merck™). Next day, membranes were stained with Revert ™ 700 Total Protein Stain (Li-Cor) for total protein measurement, blocked with Intercept (PBS) Blocking Buffer (Li-Cor) for 2 h and incubated overnight at 4 °C with the primary antibodies (1/200 anti-dystrophin antibody Abcam15277 or 1/50 anti-utrophin antibody Mancho 7). After washing steps with PBS-Tween 0.1%, membranes were incubated with secondary antibodies for 1 h (1/5000 IRDye 800CW goat anti-rabbit 926-32211 or IRDye 800CW goat anti-mouse 926-32210, Li-Cor) at room temperature, washed again with PBS-Tween 0.1% and scanned using an Odyssey Clx imaging system. Quantification of bands was performed using Image Studio ™ software.

Western blotting was performed as previously described (Schneider et al. 2017 (link)). Briefly, cells were lysed using Triton-X-100 sample buffer, and proteins were separated by SDS-PAGE. Proteins were blotted on a nitrocellulose membrane (Thermo Scientific). Detection occurred by using specific antibodies against β-actin (1:2500 dilution, Sigma-Aldrich, Munich, Germany), ACE2 and TMPRSS2 (both 1:1000 dilution, abcam, Cambridge, UK) followed by incubation with IRDye-labeled secondary antibodies (LI-COR Biotechnology, IRDye^®800CW Goat anti-Rabbit, 926-32211, 1:40 000) according to the manufacturer’s instructions. Protein bands were visualized by laser-induced fluorescence using infrared scanner for protein quantification (Odyssey, Li-Cor Biosciences, Lincoln, NE, USA).