Miracle star western blot detection system

Proteins (GSCs or tumors) were extracted with RIPA buffer in the presence of complete protease inhibitors (Roche), separated by electrophoresis, transferred to PVDF membranes (Millipore), and blocked with 5% skim milk (BD). The membranes were incubated with primary antibodies against IGFBP5 (1:200, Santa Cruz), HER2 (1:500, Cell Signaling Technology), pHER2 Y1248 (1:500, R&D System), ROR1 (1:500, Cell Signaling Technology), pROR1 Tyr786 (1:500, Thermo Fisher), CREB (48H2) (1:500, Cell Signaling Technology), pCREB Ser133 (1:500, Cell Signaling Technology), IGF1R (1:1000, Sangon Biotech), pIGF1R Tyr1165/1166 (1:1000, Sangon Biotech), GAPDH (1:1000, Cell Signaling Technology), and β-actin (1:1000, Santa Cruz) overnight at 4 °C. The immunoreactive bands were visualized using peroxidase-labeled affinity-purified secondary antibodies (KPL) and the Amersham ECL Prime Western Blotting detection reagent (GE Healthcare) or Miracle-Star Western Blot detection system (iNtRON Biotechnology).

Cell lysates were prepared using RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing protease inhibitors (Sigma-Aldrich; St. Louis, MO, USA). Protein samples were then separated by SDS-PAGE, transferred to PVDF membranes, and subsequently incubated with primary antibodies and HRP-conjugated secondary antibodies (Bio-Rad; Hercules, CA, USA). The protein bands were visualized using the Miracle-Star Western Blot Detection System from iNtRON Biotechnology (Seongnam, Korea). The following antibodies were used: Frizzled-6 (dilution 1:1000, #5158, Cell Signaling Technology; Danvers, MA, USA) and β-actin (dilution 1:5000, #BS6007M, Bioworld Technology; St. Louis Park, MN, USA).

Cells were washed with PBS and lysed with 2× sample buffer (12.5 mM Tris-HCl (pH 6.8), 2% glycerol, 0.4% sodium dodecyl sulfate (SDS), 1% β-mercaptoethanol, and 0.01% (w/v) Bromophenol Blue). Lysates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Amersham Hybond™ sequencing 0.2 μm PVDF membranes (GE Healthcare, Little Chalfont, UK), and blotted with antibodies against ZNF143 (sc-100983; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Beclin1 (#3738; Cell Signaling Technology, Danvers, MA, USA), ATG5 (#2630; Cell Signaling Technology), ATG12-ATG5 (#2630; Cell Signaling Technology), free ATG12 (#2010; Cell Signaling Technology), LC3B (#2775; Cell Signaling Technology), p62 (610832; BD Biosciences), p53 (sc-126; Santa Cruz Biotechnology), p14ARF (sc-8613; Santa Cruz Biotechnology), p-AKTSer473 (#4060; Cell Signaling Technology), p-AKTThr308 (#2965; Cell Signaling Technology), AKT (#9272; Cell Signaling Technology), and β-actin (sc-69879; Santa Cruz Biotechnology). Immunoreactivity was detected using the Miracle-Star™ western blot detection system (iNtRON Biotechnology, Jungwon, Republic of Korea).