American Society of Hematology

Figure 1

$Figure 1. Tandem affinity purification of the Rab27a/Slp2a complex. (A) TAP-Rab27aT (an inactive mutant) and TAP-Rab27aQ (an active mutant) were purified from 2 stable YT cell lines expressing these constructs. Two successive affinity purification steps were performed using IgG beads and then calmodulin beads. Total lysates, the IgG bead fraction, and the calmodulin bead fraction were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto an Immobilon membrane. Western blot analysis of TAP-tagged Rab27a mutants was revealed with anti-Rab27a. (B) A fraction of protein purified with TAP-Rab27aT and TAP-Rab27aQ after the TAP procedure was separated using 4% to 20% SDS-PAGE and visualized by Coomassie blue staining. (C) Schematic representation of the Slp2a domains used to localize the peptides identified by mass spectrometry. The peptide sequences are shown.$

Tandem affinity purification of the Rab27a/Slp2a complex. (A) TAP-Rab27aT (an inactive mutant) and TAP-Rab27aQ (an active mutant) were purified from 2 stable YT cell lines expressing these constructs. Two successive affinity purification steps were performed using IgG beads and then calmodulin beads. Total lysates, the IgG bead fraction, and the calmodulin bead fraction were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto an Immobilon membrane. Western blot analysis of TAP-tagged Rab27a mutants was revealed with anti-Rab27a. (B) A fraction of protein purified with TAP-Rab27aT and TAP-Rab27aQ after the TAP procedure was separated using 4% to 20% SDS-PAGE and visualized by Coomassie blue staining. (C) Schematic representation of the Slp2a domains used to localize the peptides identified by mass spectrometry. The peptide sequences are shown.

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