Immobilon classico or forte substrate

Cultured cells were lysed with a 1×sample buffer. Conditioned medium, cultured medium and the samples lysed with TBS buffer were mixed with a 2×sample buffer and boiled for 5 min. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (10600001; Cytiva). The following antibodies were used: anti-ß-actin (C-15) mouse monoclonal antibody (mAb) (A5441; Merck), MK (A-9) mouse mAb (sc-46701; Santa Cruz Biotechnology), RPS6 (5G10) rabbit mAb (#2217; CST), phospho-RPS6 (D57.2.2E) XP rabbit mAb (#4858; CST), insulin-like growth factor binding protein 2 (IGFBP2) (EPR18012-257) rabbit mAb (ab188200; Abcam). ALK (C26G7) rabbit mAb (#3333; CST), ApoER2 (EPR3326) rabbit mAb (ab108208; Abcam), Syndecan-2 rabbit antibody (#36-6200; Invitrogen), Horseradish Peroxidase-conjugated streptavidin (434323; Invitrogen) and peroxidase-conjugated rabbit anti-mouse IgG or goat anti-rabbit IgG (315-035-048 or 111-035-144; Jackson ImmunoResearch). Chemiluminescence was developed using the Immobilon Classico or Forte substrate (WBLUC0100 or WBLUF0100; Merck) and detected on the Amersham Imager 680 (Cytiva). Silver staining was conducted using PierceTM Silver Stain Kit (24612; Thermo Fisher Scientific Inc. Inc.) according to manufactures protocol.

Cultured cells were lysed with RIPA buffer (25 mmol/L Tris‐Cl pH 7.6, 150 mmol/L NaCl, 1% NP‐40, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (04080‐24; Nacalai Tesque). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (23225; Thermo Fisher Scientific). Whole cell extracts were mixed with 2× sample buffer (125 mmol/L Tris‐Cl pH 6.8, 4% SDS, 20% glycerol, 3.1% DTT, and 0.02% bromophenol blue) and heated for 5 minutes at 95°C. Whole cell extracts containing equal amounts of protein were separated by SDS‐PAGE and transferred to PVDF membranes (10600023; Cytiva). The following Abs were used: monoclonal anti‐ß‐actin clone C‐15 (A5441; Merck), YAP (D8H1X) XP rabbit mAb (#14074; Cell Signaling Technology), GATA‐3 (D13C9) XP Rabbit mAb (#5852; Cell Signaling Technology), Phox2a (37K‐2) (sc‐81978; Santa Cruz Biotechnology), Phox2b (B‐11) (sc‐376997; Santa Cruz Biotechnology), and peroxidase‐conjugated rabbit anti‐mouse IgG or goat anti‐rabbit IgG (315‐035‐048 or 111‐035‐144; Jackson ImmunoResearch). Chemiluminescence was developed using the Immobilon Classico or Forte substrate (WBLUC0100 or WBLUF0100; Merck) and detected on the Amersham Imager 680 (Cytiva).