storage phosphorimager screen  (Molecular Dynamics Inc)

Journal: mBio

Article Title: Lysophospholipid signaling coordinates outer membrane homeostasis in Escherichia coli

doi: 10.1128/mbio.00567-26

Figure Lengend Snippet: PF resistance and reduced envelope stress in LPL-recycling mutants are PldA-dependent. ( A ) Efficiency of plating assays comparing WT and LPL-recycling mutants lacking PldA in the absence or presence of 25 ng/mL PF. ( B ) LPS profiles of WT and ∆ pldA strains grown with PF. Cultures were grown to an OD 600 of 0.4 and exposed to 40 ng/mL PF for 4 h, and changes in LPS were assessed by SDS-PAGE. Capsule addition is indicated by a bracket. ( C ) TLC of extracted GPL species from WT or ∆ pldA backgrounds with (red) and without (black) PF treatment. Following radiolabeling, GPLs were isolated and separated in the solvent chloroform:methanol:acetic acid (65:25:10, vol/vol/vol). Lipids (20,000 cpm per lane) were visualized by phosphorimaging, and densitometry within each lane was used to determine the percentage of lyso-PE present. Changes in lipid composition were similar for other biological replicates. Data are representative of a minimum of three biological replicates.

Article Snippet: Plates were exposed to a phosphorimaging screen overnight and imaged using the Amersham Typhoon Biomolecular Imager (Cytiva).

Techniques: SDS Page, Radioactivity, Isolation, Solvent

Journal: mBio

Article Title: Lysophospholipid signaling coordinates outer membrane homeostasis in Escherichia coli

doi: 10.1128/mbio.00567-26

Figure Lengend Snippet: Loss of LPL recycling improves OM asymmetry during LpxC inhibition. ( A ) TLC of lipid A species from WT and ∆ pldA backgrounds grown with (red) and without (black) PF. Each sample (5,000 cpms) was separated in the solvent chloroform:pyridine:88% formic acid:water (50:50:16:5, vol/vol/vol/vol). Lipids were visualized by phosphorimaging, and densitometry was used to calculate the percentage of hepta-acylated lipid A (i.e., PagP activity). Changes in lipid composition were similar for other biological replicates. ( B ) GPL-to-LPS ratios in the absence and presence of PF. Cultures were grown to an OD 600 of 0.4 and exposed to 40 ng/mL PF for 2 h. GPLs and lipid A were extracted and quantified by scintillation counting. ( C ) Quantification of GPL and lipid A production following PF treatment. The cpms for each lipid sample were normalized to the total amount of 32 P incorporation into the cell. GPL and lipid A levels relative to replicate 1 of WT are shown. Data for GPL-to-LPS ratios is from five biological replicates. ( D ) GPL synthesis assay during LpxC inhibition. Cells were grown with 32 P i to an OD 600 of 0.4, when 40 ng/mL PF was spiked into cultures. At 30-min intervals over a 2 h period, aliquots were taken for GPL extraction and for the determination of total cellular 32 P incorporation. Counts from extracted GPLs were first normalized to total radiolabel incorporation and then compared to WT GPL levels at 0 min and plotted over time. Data are representative of three biological replicates. ( E ) Efficiency of plating assays assessing changes in OM permeability. Cultures were grown to an OD 600 of 0.4, treated with 30 ng/mL PF for 2 h, washed, and normalized by OD 600 . Serial dilutions were spotted on the indicated LB agar plates, and growth was evaluated. Error bars represent SD for bar graphs (** P -value ≤ 0.01; **** P -value < 0.0001). Data are representative of a minimum of three biological replicates.

Article Snippet: Plates were exposed to a phosphorimaging screen overnight and imaged using the Amersham Typhoon Biomolecular Imager (Cytiva).

Techniques: Inhibition, Solvent, Activity Assay, Extraction, Permeability