Anti phospho rps6kb1 antibodies

Differentiated Dictyostelium cells were incubated in DB with 2 mM caffeine for 20 min at 22°C, washed twice with ice-cold DB, and resuspended at 5 × 10⁷ cells/ml (Senoo et al., 2019 (link)). Cells were shaken at 200 rpm at 22°C and stimulated with 1 μM cAMP (Kamimura et al., 2009 (link); Senoo et al., 2019 (link)). Aliquots were taken at different time points and lysed in SDS-PAGE sample buffer. Phosphorylation of PkbA and PkbR1 were detected using anti-phospho-RPS6KB1 antibodies (Cell Signaling, 9205) because of the conserved amino acid sequences around the phosphorylation sites (Senoo et al., 2019 (link)). Phosphorylation of Erk2 was detected using anti-phospho-p44/42 MAPK antibodies (Cell Signaling, 4370), as previously described (Nichols et al., 2019 (link)).

Differentiated Dictyostelium cells were incubated in DB with 2 mM caffeine for 20 min at 22°C, washed twice with ice-cold DB, and resuspended at 5×10⁷ cells/ml. Cells were shaken at 200 rpm at 22°C and stimulated with 1 μM cAMP ^{39 (link)}. Aliquots were taken at different time points and lysed in SDS-PAGE sample buffer. Proteins were analyzed by immunoblotting with antibodies to PKBA and PKBR1. As extensively described in many studies ^{23 (link)}, phosphorylation of PKBA and PKBR1 were detected using anti-phospho-RPS6KB1 antibodies (Cell Signaling, 9205) and anti-phospho-PKC antibodies (Cell Signaling, 2060) because of the similar amino acid sequences around the phosphorylation sites (Fig. S4).