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Fig. 3 | BMC Microbiology

Fig. 3

From: Legionella effector AnkX interacts with host nuclear protein PLEKHN1

Fig. 3

AnkX interacts with PLEKHN1 directly. a, b Reciprocal pull-downs were performed with MagneHaloTag or MagneGST beads and lysates from E. coli producing GST-AnkX or HaloTag-PLEKHN1. Inputs (30 μg) and eluates (43%) were analyzed via SDS-PAGE and immunoblot analysis. Anti-GST and anti-HaloTag antibodies were used to determine that GST-AnkX was captured when HaloTag-PLEKHN1, but not when HaloTag, was present on the beads. The reverse pull-down supported this result, showing that HaloTag-PLEKHN1 was precipitated with GST-AnkX, but not GST. Estimated molecular weights: HaloTag (34 kDa), HaloTag-AnkX (141 kDa), HaloTag-PLEKHN1 (106 kDa), GST (26 kDa), GST-AnkX (133 kDa), GST-PLEKHN1 (98 kDa). c GFP and GFP-tagged PLEKHN1 were immunoprecipitated from transiently transfected HEK293T cells using GFP-Trap™ MA beads, and incubated with lysate of HEK293T cells transiently producing HaloTag-AnkX. Inputs (~2.8%) and eluates (~60%) were separated by SDS-PAGE, and AnkX was detected by immunoblot using anti-HaloTag antibody. The bottom panel shows the GFP and GFP-PLEKHN1 present on the beads. Estimated molecular weights: HaloTag-AnkX (141 kDa), GFP (27 kDa), GFP-PLEKHN1 (94 kDa)

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