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.
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.