Additional file 1: Table S1. of Legionella effector AnkX interacts with host nuclear protein PLEKHN1

Microbial strains and plasmids used in this study. Table S2. List of oligonucleotides used for this study. Figure S1. Quality of NAPPA arrays in protein production. (A) Representative images of DNA PicoGreen and GST staining before and after in vitro DNA transcription and translation. (B) Distribution of fluorescent signal intensity of proteins on NAPPA microarrays. The expression rate of proteins that were displayed on NAPPA was calculated by using the signals of nonspots (buffer) plus two standard deviations. Figure S2. Correlation of NAPPA protein microarrays. The array contains 2206 human genes. The GST-proteins displayed on NAPPA were detected by mouse anti-GST antibody followed by HRP labeled goat anti-GST secondary antibody. Figure S3. Workflow of bead-based pull-down assay used in the validation of protein-protein interactions. Figure S4. Workflow of wNAPPA approach used for the validation of protein-protein interactions. Figure S5. Increasing SDS concentrations disrupt AnkX dimer formation. HEK293T cells ectopically producing HaloTag-AnkX were lysed and the post-nuclear supernatant (PNS) was collected. The PNS was incubated with increasing amounts of SDS (1.38, 1.72, 2.05, and 2.38%) in Laemmli buffer for 5 min at 80 °C. Figure S6. Representative SR-SIM maximum-intensity projection image displaying immunofluorescence of endogenous PLEKHN1. The subcellular distribution of PLEKHN1 is predominantly nuclear. PLEKHN1 is also found as puncta dispersed throughout the cytosol and a larger vesicular structure. Scale bar: 10 μm. Figure S7. PLEKHN1 interaction candidates revealed by wNAPPA. PLEKHN1 fused with a C-terminal HaloTag were co-produced with their interaction proteins using human cell-free expression system. The resulting protein complexes were captured by an anti-GST antibody-coated ELISA plate, and retention of AnkX-HaloTag was detected immunologically. These interaction proteins were selected based on the signal-to-noise ratio above 3. The HaloTag was used as a negative control. The Rab35 and LidA were employed as a positive control. (DOCX 3407 kb)