JCS032763 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1 Representative blots showing the effect of p110 inactivation on Akt phosphorylation. The indicated BMMs were stimulated with 30 ng/ml CSF1 for different time points, followed by analysis of phosphorylation of Akt (on T308 and S473) or Erk1/2 (on T202/Y204) by western blot of total cell lysates (80 µg/lane). (A) Pharmacological inactivation of p110α, p110β or p110δ. (B) Heterozygous inactivation of p110δ (left panel) or p110α (right panel).

• Supplemental Figure S2 -

  Fig. S2 Representative blots showing the effect of p110 inactivation on GTP-loading of Rac1 and RhoA. (A) Cells were pre-treated with the p110 inhibitors as indicated and then equal volumes of cell lysates from untreated or CSF1-treated cells were incubated with GTP-PBD bound to glutathione-agarose, resolved by SDS-PAGE and immunoblotted for coprecipitated Rac1. Total cell lysates were resolved on the same SDS-PAGE gel and immunoblotted for Rac1. (B) The indicated BMMs were stimulated with 30 ng/ml CSF1 for different time points, followed by determination of Rac1-GTP levels. (C) Cells were pre-treated with the p110 inhibitors as indicated, followed by incubation of equal volumes of lysates from cells (stimulated with or without CSF1) with GST-RBD to affinity precipitate GTP-bound RhoA. Total cell lysates were resolved on the same SDS-PAGE gel and immunoblotted for RhoA. (D) The indicated BMMs were stimulated with 30 ng/ml CSF1 for different time points, followed by determination of RhoA-GTP levels. (E) Cre(-) or Cre(+) BMMs were stimulated with 30 ng/ml CSF1 for different time points, followed by determination of RhoA-GTP levels.

• Supplemental Figure S3 -

  Fig. S3 Effect of genetic inactivation of p110δ or p110α on DNA synthesis of BMMs. The indicated BMMs were incubated with different concentrations of CSF1 and ³HThymidine incorporation was measured as described.

• Supplemental Figure S4 -

  Fig. S4 Representative blots to show the effect of isoform-selective PI3K inactivation on Akt phosphorylation in macrophage cell lines. The indicated cells were stimulated with 30 ng/ml CSF1 for different time points, followed by analysis of phosphorylation of Akt (on T308 and S473) by western blot of total cell lysates (80 µg/lane).