Cold cell lysis buffer

Cells were lysed with cold cell lysis buffer (Cell Signalling Technology) with protease inhibitor (Roche) and phosphatase inhibitor (Roche). For preparation of cytoplasmic and nuclear protein fractions, cells were lysed as described previously, left on ice for 15 minutes and spun for 10 minutes at 13000rpm. The supernatant (cytoplasmic fraction) was transferred to a new tube and the remaining pellet was washed with PBS and lysed with 1x nuclear lysis buffer (50 mM Tris-HCL (pH 8.1); 5 mM EDTA; 1% SDS) (nuclear fraction). The determination of cellular protein was done in accordance to a series of BSA standards using the Protein Assay kit (Bio-Rad) based on the Lowry assay [57 (link)]. Proteins were run in 10–15% acrylamide gel (Severn Biotech) then transferred to a Immobilon-P PVDF membrane (Millipore) for probing with primary antibodies (listed in Supplementary Table 3).
Immunohistochemistry was performed with a Leica Microsystem Bond III automated machine using the Bond Polymer Refine Detection Kit (Ref DS9800) followed by Bond Dab Enhancer (AR9432). The slides were dewaxed with Bond Dewax Solution (AR9222). Heat mediated antigen retrieval was performed using Bond Epitope Retrieval Solution for 10 mins. Primary antibody dilution for anti-LGR5 (Proteintech 21833-1-AP) was1:400.

The human lung carcinoma cell lines, H226, H460, H520, and A549 (2 × 10⁵/well) were seeded in six‐well tissue culture plates and incubated for 24 hours. The cells were left untreated or treated with IFN‐γ (10 or 50 ng/mL), IFN‐α (800 U/mL), or IFN‐β (10 ng/mL) and/or ODNs (3 μM), and further cultured for 30 minutes or 16 hours. Then, the cells were lysed in cold cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease and phosphatase inhibitors (Cell Signaling Technology) and centrifuged for 10 minutes at 14 000 × g at 4°C. Samples containing 30 μg protein were boiled for five minutes, size‐separated on a 10% precast gel (Bio‐Rad, CA, USA), and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific Inc.). The immunoblots were probed with antibodies specific for JAK1, phosphorylated (p)‐JAK1, JAK2, p‐JAK2, STAT1, p‐STAT1, PD‐L1, β2‐MG, indoleamine 2,3‐dioxygenase 1 (IDO), and β‐actin, followed by probing with anti‐rabbit IgG horseradish peroxidase (HRP)‐linked secondary antibody (Cell Signaling Technology). The signals were visualized with Image Quant LAS 500 (GE Healthcare UK Ltd., Buckinghamshire, England).