Adi sab 300

Gefitinib (AstraZeneca), Phenylarsine Oxide (PAO) (Sigma‐Aldrich), Filipin III (Sigma‐Aldrich), anti‐EGFR (sc‐373746), anti‐EGFR (4267, Cell signaling), anti‐p‐EGFR (2234, Cell signaling), anti‐STAT3 (sc‐8019), anti‐p‐STAT3 (sc‐8059), anti‐ERK (9102, Cell signaling), anti‐p‐ERK (9101, Cell signaling), anti‐EEA1 (610457, BD Biosciences), anti‐PARP (9542, Cell signaling), anti‐c‐Myc (9402, Cell signaling), anti‐β‐actin (A5316), Goat anti‐mouse IgG (H + L)‐HRP conjugate (1706516, Bio‐Rad), Goat anti‐rabbit IgG polyclonal HRP conjugated (ADI‐SAB‐300, Enzo), Alexa Fluor 488 goat anti‐rabbit antibody (A32731, Thermo Fisher Scientific), and Alexa Fluor 594 goat anti‐mouse antibody (A32742, Thermo Fisher Scientific).

The mRNAs were injected at the one-cell stage of embryos and collected at stage 11 for western blots. Non-injected embryos served as the negative control. Collected embryos were lysed in lysis buffer with phosphatase and protease inhibitors, in preparation for resolving the proteins with 10% SDS-PAGE and transfer to a PVDF membrane, with the membrane blocked and incubated with either pSmad1 pSer-463/465 (CS-9511S) or pSmad1 pSer-206 (CS-9553P) antibodies. Following washes of the PVDF membrane, it was incubated with the enzyme-labeled secondary antibody (ADI-SAB-300, Enzo Biochem, Farmingdale, NY). The protein signals were visualized by an ECL detection kit (GE Healthcare, Chicago, IL).

Cell lysates were prepared to detect the protein expression of cleaved PARP1. Equal amounts of protein (25 μg) were boiled with Laemmli sample buffer (Bio-Rad, USA) for 5 min and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels. Separated proteins were then transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, USA) and blocked with 5% skim milk (Millipore) in Tris-buffered saline (TBS) containing 0.01% Tween-20 (TBST) for 30 min. The membranes were washed three times for 30 min in TBST and incubated with PARP1 (1:1,000 dilution, v/v, Cat#9542, Cell Signaling Technology, USA) and β-actin (1:2,000 dilution, v/v, Cat#sc-47778, Santa Cruz Biotechnology, USA) antibody in TBST buffer at 4°C overnight. The membranes were washed once every 10 min for 1 h in TBST and then incubated with secondary anti-rabbit (1:2,000 dilution, v/v, Cat#ADI-SAB-300, Enzo Life Sciences, USA) and anti-mouse (1:2,000 dilution, v/v, Cat#ADI-SAB-100, Enzo Life Sciences) HRP-conjugated antibodies for 1 h in TBST at room temperature. Primary and secondary antibodies were diluted with TBST and washed once every 10 min for 1 h. For visualization of the blots, the membranes were developed using D-Plus ECL Pico solution (Dongin Biotech, Korea).