Journal: EMBO Reports
Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol
doi: 10.1038/s44319-024-00328-x
Figure Lengend Snippet: ( A ) Immunoblot analysis of SseI-interacting proteins (IQGAP, TRIP6, and ARF1) on isolated peroxisomes from STM-infected (MOI = 10) and uninfected HeLa cells, with whole cell lysate (WCL). The representative blot was probed with anti-IQGAP, anti-TRIP6, and anti-ARF1 antibodies. ( B ) Immunoblot analysis of proteins after SseI pulldown from HeLa cells expressing HA-tagged SseI WT and SseI ΔGKM. Cell lysates (input) were used for SseI pulldown with anti-HA antibody. IgG was used as a control. The representative blot was probed with anti-HA, anti-ARF1, and anti-PEX5 antibodies. ( C ). Immunoblot analysis of in vitro binding between purified His-tagged SseI and HA-tagged ARF1 in HeLa whole cell lysates (WCL). The representative blot was probed with anti-His and anti-HA antibodies. ( D ) Immunoblot analysis of Active ARF1 was pulled down from WT, Δ sseI STM-infected (MOI = 10), and uninfected HeLa cells. Immunoblot analysis was performed to detect active ARF1. Ponceau staining was used as a loading control for the pulldown samples. Total ARF1 levels in whole cell lysates were assessed by immunoblotting, with actin as a loading control. ( E ) Densitometry analysis of ARF1 activation in WT and Δ sseI STM (MOI = 10) infected HeLa cells relative to uninfected controls. Data represent the mean ± SEM of three independent experiments. ( F ) Immunoblot and densitometry analysis of Active ARF1 protein levels after ARF1-GTP pulldown from isolated peroxisomes, cells infected with WT or Δ sseI STM, or Δ sseI STM complemented with pQE60-SseI clones (ΔGKM, SKL, full length (FL) (MOI = 10). Densitometry analysis represents three independent experiments. ( G ) Fold proliferation of WT or Δ sseI STM (MOI = 10) in HeLa cells transfected with full-length or constitutively active (Q71L) ARF1 or silenced ARF1. Data represent the mean ± SEM of three independent experiments. ( H ) ELISA analysis of PI (4,5) P2 (PIP2) levels on isolated peroxisomes from PEROXO-tag expressing HeLa cells infected with WT or Δ sseI STM. Data from three independent experiments are shown. ( I , J ) Fold proliferation of STM in HeLa cells after silencing PIP4K2A ( I ) or PIP4K2B ( J ). Data represent the mean ± SEM of three independent experiments. ( K ) Fold proliferation of WT STM (MOI = 10) in HeLa cells treated with PIP4K inhibitor THZ-P1-2 (1 µM). Data represent the mean ± SEM of three independent experiments. ( L , M ) Fold proliferation of WT STM (MOI = 10) in human monocyte-derived dendritic cells (Mo-DCs) ( L ) and human monocyte-derived macrophages (MDMs) ( M ) treated with PIP4K inhibitor THZ-P1-2 (1 µM). Data are from four healthy human donors. Data information: Data were analyzed using one-way ANOVA (Sidak’s multiple comparisons test), ( E ) (* p = 0.0211, * p = 0.0138), ( F ) (** p = 0.0092, ** p = 0.0058), ( G ) (**** p < 0.0001), and ( H ) (**** p < 0.0001), ‘ns’ denotes no significant difference. ( I – M ) Data were analyzed using student’s t -test, ( I ) (** p = 0.0091), ( J , K ) (** p = 0.0046), ( L ) (*** p = 0.0002), ( M ) ((*** p = 0.0003). ‘ns’ denotes no significant difference. .
Article Snippet: Phosphatidylinositol (4,5) bisphosphate mass ELISA assay The PI(4,5)P2 levels extracted from peroxisomes were measured using a competitive ELISA assay (Echelon Biosciences Inc, K-4500).
Techniques: Western Blot, Isolation, Infection, Expressing, Control, In Vitro, Binding Assay, Purification, Staining, Activation Assay, Clone Assay, Transfection, Enzyme-linked Immunosorbent Assay, Derivative Assay
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