Validation and retroviral expression of Pak2 biochemical mutants in HSPCs. (A) Schematic of polycistronic MSCV retroviral vectors expressing Cre and WT Pak2 or mutants with EGFP. (B) PCR analysis of genomic DNA extracted from FACS-sorted EGFP⁺ LSK cells harvested from Pak2^fl/fl mice that were transduced with the indicated vectors; untransduced cells were used as a control. The positions of the WT and deleted bands are shown on the left and size ladder is shown on right. (C) Immunoblot to detect Pak2 expression in FACS-sorted EGFP⁺ LSK from WT (control) or Pak2^fl/fl mice transduced with Cre alone (Pak2-deleted, Pak2^Δ/Δ) or Cre-T2A-Pak2-WT (Pak2-WT). β-actin is used as a loading control. (D) In vitro kinase assay using MBP as a substrate and HA-tag immunoprecipitates from 32D cells transduced with vectors expressing Pak2-WT or Pak2 mutants. The blot showing phosphorylated threonine (p-Thr) and serine (p-Ser) residues and MBP indicates presence of substrate in all conditions. Bottom panel shows exogenous (HA) and total Pak2 expression from cell lysates with β-actin as a loading control. (E) Coimmunoprecipitates of HA-tagged Pak2-WT, Pak2-KD, and Pak2-Δβ-Pix from transduced 32D cell lysates analyzed by immunoblot for β-Pix. β-Pix expression in total cell lysates and HA blot of immunoprecipitates were used as loading controls. Ψ, Ψ packaging signal; EGFP, enhanced green fluorescent protein; IB, immunoblot; IP, immunoprecipitation; LTR, long terminal repeat; TCL, total cell lysate.