Hyperfilm

Whole‐cell extracts were prepared by scraping cells using 1× PBS and cells were lysed for 25 min in whole‐cell lysis buffer (10 mmol/L Tris‐HCl pH 8.0, 420 mmol/L NaCl, 250 mmol/L sucrose, 2 mmol/L MgCl₂, 1 mmol/L CaCl₂, 1% Triton‐X100) supplemented with complete protease inhibitor cocktail (Roche, Mississauga, Canada, 04 693 116 001), and then centrifuged at 15,000 g for 10 min at 4 °C. Supernatants were collected and protein concentrations were determined using a protein assay kit (Bio‐Rad, 500‐0006). Thirty micrograms of total protein lysate were separated by electrophoresis on Criterion XT Bis-Tris 4-12% (345-0125) and transferred to a polyvinylidene difluoride membrane (Millipore). Blots were blocked using 5% milk in TBST (500 mmol/L Tris pH 7.6, 2 mol/L NaCl, 0.5% Tween) at room temperature for at least 1 h and then incubated overnight at 4 °C with primary antibodies specific for RAB27A (1:200; Abcam ab55667) or ACTIN (1:50,000; Sigma A5441) or Horseradish‐peroxidase (HRP)‐conjugated antibodies were used for 1 h at room temperature. A goat anti-mouse secondary (1:30,000; Thermo Scientific, 31430) was used to detect ACTIN and RAB27A; The antigen‐antibody complexes were visualized using the chemiluminescent HRP substrate (Millipore, WBKLS0500) following the manufacturer’s instructions and exposed to Hyperfilm (GE Healthcare, Baie-d’Urfé, QC, Canada, 28‐9068‐35).

Proteins were isolated using a modified RIPA lysis buffer containing 50 mM Tris HCl, 1% NP-40, 1% natrium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄ and 1x Protease inhibitor cocktail (Roche). Equal amounts of protein were electrophoresed through 4–12% Bis/Tris gels (Life Technologies), transferred to Hybond-P membranes (GE Healthcare Biosciences, Pittsburgh, PA, USA), and immunodetected with rat anti-E2A (clone# 826927, clone R&D Systems, Minneappolis, MN), or rabbit anti-GAPDH (Cat. # G9545, Sigma) antibodies. Bands were detected by chemiluminescence using ECL Plus Western Blotting Detection System and HyperFilm (GE Healthcare Biosciences). Quantification by densitometry was performed using Image J Software [27 (link)].

Equal amount of proteins (50 μg) from three biological replicates were separated on a 12% SDS-PAGE gel and. The uniformity of the protein amount loaded on the gels was also visulized by staining SDS-PAGE gels using Coomassie Brilliant Blue (Fig. S3). For Western blot analysis, proteins were electrophoretically transferred onto polyvinylidine difluoride membranes (Bio-Rad). The blots were blocked with TBST (20 mM tris-HCl, pH 7.6, 150 mM NaCl, and 0.1%ween-20), containing 5% BSA, followed by incubation with primary antibody solution overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Signals were detected with ECL substrate (GE) using Hyperfilm (GE). Supplemental Table S3 represents the primary and secondary antibodies used. Gapdh was used as the loading control. Protein bands were quantified by using ImageJ software. The volume of each band was analyzed using student’s t-test.

Western blot analysis was carried out on whole cell extracts (50 μg) upon various treatments, after fractionation by PAGE gel electrophoresis and transferred to PVDF membranes for immunoblotting with antibodies listed in Supplementary Table 3. HRP-conjugated secondary antibodies against rabbit and mouse were all obtained from Cell Signalling Tech. The ECL system (Clarity ECL substrate, Biorad) and hyperfilm (GE Healthcare, Munich, Germany) were used for membrane visualization.

Gel mobility shift assays were performed using a labeled probe containing either native or mutagenized fprA-promoters amplified from pP_fprA, pP_fprA-MU1, or pP_fprA-MU2 as a template and using ³²P-labeled BT4691 and BT4692 primers. The promoter fragments (EBI61and EBI62) with and without proposed FinR binding site were amplified from genomic PAO1 using primers, EBI61-EBI70 and EBI62-EBI69, respectively. Binding reactions were conducted using 3 fmol of labeled probe in 25 μl of reaction buffer containing 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 4 mM MgCl₂, 0.5 mM EDTA, 0.02 mg ml^-1 bovine serum albumin (BSA), 5 mM dithiothreitol (DTT), 10% (v/v) glycerol, and 200 ng of poly(dI-dC). Various amounts of purified FinR were added, and the reaction mixture was incubated at 25°C for 20 min. Protein-DNA complexes were separated by electrophoresis on a 5% non-denaturing polyacrylamide gel in 0.5× Tris-borate-EDTA buffer at 4°C and were visualized by exposure to Hyperfilm (GE Healthcare).