Ab49969

Antibodies against the following proteins were obtained from the indicated suppliers: γH2AX (Millipore, 05–636; and CST, 9718), β-actin (Sigma, AC-74), 53BP1 (Merck, PC712), APOBEC3B (GeneTex, GTX17214), and HA-tag (Abcam, ab49969). Western blotting was performed as described previously [15] (link). Immunostaining was also performed as described previously [16] (link) using a confocal laser microscope (Olympus, FV10i). Before immunostaining with primary and secondary antibodies, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100/PBS for 10 min. For confocal microscope imaging, cells were cultured on coverslips and stained as above.

Cells were harvested at the indicated times and rinsed twice with PBS. Cell extracts were prepared with lysis buffer and centrifuged at 13,000 g for 10 min at 4 °C. Protein samples (100 μg) were electrophoresed using 10 % polyacrylamide gels and transferred to PVDF membranes. After the membranes were blocked with 5 % BSA for 1 h at room temperature, they were incubated with FOXD3 antibody (ab67758, 1:1,000, Abcam, Cambridge, MA, USA), STAT3 antibody (#4904, 1:2,000, Cell Signaling, Danvers, MA, USA), p-STAT3(Tyr705) antibody (sc-8059, 1:1,000, Santa Cruz, Dallas, TX, USA), p-STAT3(Ser727) antibody (sc-293059, 1:1,000, Santa Cruz, Dallas, TX, USA), Flag antibody (ab49969, 1:3,000, Abcam, Cambridge, MA, USA) or HA antibody (ab18230, 1:3,000, Abcam, Cambridge, MA, USA) in 5 % BSA overnight at 4 °C. Secondary antibodies (1:3,000) were labeled with horseradish peroxidase (HRP). The signals were checked by autoradiography film when HRP substrate was added to the membranes.

At 24 h post transfection, the cells were collected and seeded onto a six-well plate. After 48 h, the cells were lysed and sonicated. Then, protein concentrations of lysates were measured using a Protein Assay Kit (Bio-Rad, Hercules, CA). The lysates were re-suspended in Laemmli buffer, denatured for 5 min at 98°C, and separated by Tris-glycine denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were blotted onto polyvinylidene fluoride membranes, blocked in 5% milk, and incubated overnight with the following primary antibodies: for endogenous protein detection, anti-E-cadherin (ab40772; Abcam, Cambridge, United Kingdom) or anti-α-tubulin (ab11304; Abcam); for MS2-22sTag protein detection, anti-HA (ab49969; Abcam) at a 1:1,000 (ab40772 and ab11304) or 1:2,000 (ab49969) dilution ratio in Can Get Signal Solution 1 (Toyobo) at 4°C. Subsequently, the proteins were incubated with the corresponding horseradish peroxidase–conjugated secondary antibodies (Thermo Fisher Scientific) at a 1:250 dilution ratio in Can Get Signal Solution 2 (Toyobo) for 1 h at room temperature. Chemiluminescent signals were generated using a SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) and captured on X-ray films (Fujifilm, Tokyo, Japan). The films were scanned, and signal intensities were quantified using ImageJ software.^*