Anti zbtb7a

For the metabolic labeling of cellular proteins, cells were grown in the presence of [³⁵S]-methionine and [³²P]-orthophosphate to test the protein phosphorylation, as described previously [9 (link)].
Immunoprecipitation was performed using protein A/G-presenting magnetic beads (Thermo Fisher Scientific, Darmstadt, Germany) as described before [9 (link)] using 1 to 2 mg of cell protein lysate in RIPA buffer with appropriate amounts of the following antibodies: anti-PURB (Thermo Fisher Scientific, Darmstadt, Germany): 5 µg; anti-ZBTB7A (Abcam, Cambridge, UK): 5 µg; CKbeta2 (Thermo Fisher Scientific, Darmstadt, Germany: 4.2 µg; FOXK1 (Cell Signaling Technology, Danvers, MA, USA): 3 µg; BTF3 (Abcam, Cambridge, UK): 8.5 µg; BCAR1 (OriGene Technologies GmbH, Herford, Germany): 4 µg and MAPKAPK3 (Cell Signaling Technology, Danvers, MA, USA): 6 µL (no protein concentration given by the supplier). Elution was carried out with 12 µL HCl·glycine (pH 2.5) solution. Eluates were mixed with 10 mL scintillation cocktail (UltimaGold; PerkinElmer, Waltham, MA, USA), and radioactivity was determined in a liquid scintillation counter (Tricarb 2900, PerkinElmer, Waltham, MA, USA), applying the transformed Spectral Index of the External Standard/Automatic Efficiency Control method.

Cells were harvested for Western blotting by scraping and washing with cold phosphate-buffered saline (PBS). The cells were lysed with lysis buffer for 30 min on ice. Lysates were centrifuged at 13,000 rpm for 20 min at 4 °C, and the protein concentration of the supernatant was measured using Pierce 660 nm Protein Assay Reagent (Thermo, 22660). Total cell lysate proteins were separated using sodium dodecyl sulfate‒polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes. The transferred membranes were blocked by incubation for 1.5 h in 5% w/v nonfat Difco skim milk (BD Biosciences, 232100) blocking buffer. The primary antibodies were incubated together for 3 h or overnight at 4 °C. After washing, the secondary antibody was incubated for 1.5 h. The results were visualized by the developer after Western blot analysis. The antibodies used were anti-ZBTB7A (Abcam, ab70208), anti-EPB41L5 (Thermo, PA5-58009), anti-β-actin (Sigma‒Aldrich, A5441), anti-Myc (MBL, M192-3), anti-N-cadherin (sc-59987), anti-β-catenin (sc-7963), anti-vimentin (sc-32322) (Santa Cruz Biotechnology), and anti-Snail (Cell Signaling, 3879 S). All secondary antibodies were purchased from Thermo (31430 and 31460).