Enhanced chemiluminescence reagents

Huh7 cells were treated with 100 nM 10M-D42AN for 0 to 48 h and with EGF (Invitrogen) (0–100 ng/ml) for 0 to 3 h. To observe the phosphorylation levels of Ack1 and WASP, HepG2 cells were transfected with T7-Ack1-His-pcDNA using Lipofectamine 2000 and then cultured for 48 h. These cells were lysed with RIPA buffer containing a protease inhibitor cocktail and phosphatase inhibitor cocktail. Lysates were centrifuged at 4 °C and 13,200 rpm for 15 min, and the supernatant was removed. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo). Equivalent amounts of protein were separated in NuPAGE 4% to 12% Bis–Tris gel (Thermo) or NuPAGE 3% to 8% Tris-acetate gel (Thermo) followed by incubation with primary antibodies against Ack1, EGFR, phosphorylated-EGFR Y845, pN-WASP (Santa Cruz), pAck1, GAPDH (Abcam, CST), Chk1, pChk1, phosphorylated-EGFR Y1045, Y1068, N-WASP (CST), and γH2AX (Merck) and with HRP-conjugated antibodies against FLAG, T7 (Novagen), and GST (Santa Cruz). HRP-conjugated secondary antibody against rabbit or mouse (Abcam, CST) was then applied. The blots were developed using enhanced chemiluminescence reagents (Nacalai Tesque, Cytiva).

Proteins were extracted from the hippocampus using 20 volumes of RIPA Lysis and Extraction Buffer (#89900, Thermo Fisher Scientific), containing a protease inhibitor and phosphatase inhibitor cocktails (#04080 and #07575-51, Nacalai Tesque). They were loaded onto sodium dodecyl sulfate-polyacrylamide gels for separation. The separated proteins were transferred onto 0.2-μm polyvinylidene fluoride membranes. After blocking the membrane in 5% w/v bovine serum albumin in 1 × TBST, primary antibodies against phosphorylated (p)-ERK1/2 (1:1000, #9101S; Cell Signaling Technology, Danvers, MA, USA), pan-ERK (1:1000, #610124; BD Biosciences, San Jose, CA, USA), p-CREB (1:1000, #9198; Cell Signaling Technology), CREB (1:1000, #9197S; Cell Signaling Technology), and α-tubulin (1:1000, #2144S; Cell Signaling Technology) were added at 4 °C overnight. After washing in 1 × TBST, the membrane was incubated for 1 h at room temperature with anti-rabbit or anti-mouse secondary antibodies (1:1000, #7074S; Cell Signaling Technology). Signals were detected using enhanced chemiluminescence reagents (#11644, Nacalai Tesque) and the bands were scanned using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA) following washing with TBST. A quantitative analysis was performed using ImageJ software (Version 1.53; NIH, Bethesda, MD, USA).