Cellytictm m

Sodium hydroxide was purchased from Merck (Darmstadt, Germany); ethanol, hydrochloric acid, and dimethylsulphoxide were purchased from RCI Labscan Limited (Bangkok, Thailand); diethanolamine, albumin from bovine serum, CelLytic^TM M, penicillin-streptomycin solution, and Bradford reagent were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA); magnesium chloride hexahydrate and 4-nitrophenol were purchased from Sigma-Aldrich Co. (Tokyo, Japan); 4-nitrophenyl phosphate disodium salt hexahydrate was purchased from Sigma-Aldrich, Co. (Dorset, UK); PrestoBlue^TM cell viability reagent was purchased from Life Technologies Corporation (Eugene, OR, USA); Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline pH 7.4, fetal bovine serum (FBS), MEM non-essential amino acid solution, and 0.5% trypsin-EDTA solution were purchased from Life Technologies (Paisley, UK).

PS gene deleted HTL cells were seeded at a density of 0.8 × 10⁶/well in 6-well plates and transfected the following day with 300 ng of BirA biotin ligase encoding DNA, 500 ng of Avi–C99 encoding DNA, and 500 ng of C99–TEV site–rTA–FLAG encoding DNA with Lipofectamine® 2000 (Invitrogen) transfection reagent. After transfection, biotin solution was added to 40 μm. Cells were harvested and lysed in CelLytic^TM M (Sigma-Aldrich) 1 day after transfection. The crude supernatant protein extracts were incubated with prewashed Streptavidin MagBeads (GenScript) for about 30 min, followed by three washes with lysate buffer. All samples with beads were subjected to SDS-PAGE for Western blot analysis using anti-FLAG and anti-β-actin antibodies. The β-actin level was used as loading control.

Cells were harvested and lysed in CelLyticTM-M (Sigma-Aldrich, St. Louis, MO) for 30 min on ice. Cell lysates (10 μg/lane) were separated by SDS-PAGE using SuperSepTM (Wako, Osaka, Japan) and transferred onto Polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h and then incubated with the respective primary antibody overnight at 4°C. The antibody against GPNMB was from R&D Systems (Minneapolis, MN), antibodies against HER2 and phospho-HER2 were purchased from EPITOMICS (Burlingame, CA), and all other antibodies (EGFR, phospho-EGFR, p42/44 MAPK [ERK], phospho-p42/44 Mitogen-activated protein kinase (MAPK) [ERK], and β-actin) were obtained from Cell Signaling Technology (Danvers, MA). The membranes were washed and incubated with the appropriate secondary antibodies. The immunoreactive proteins were visualized and captured by an LAS-4000 system (FUJIFILM, Tokyo, Japan).