Pip array

Phospholipid overlay assays were performed with lipid strips and PIP arrays purchased from Echelon Biosciences (Salt Lake City, UT). Nitrocellulose membranes pre-spotted with different phospholipids were blocked with 3% fat-free BSA in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% Tween 20 for 1 h at room temperature and incubated with 100 nm recombinant LtpM constructs in 2 ml of blocking buffer at 4 °C for 5 h. Binding of the proteins to lipids was visualized by immunodetection with primary anti-LtpM mouse polyclonal antibody and secondary anti-mouse IgG antibody (Table 3).

PIP Strips and PIP Arrays (Echelon Biosciences) were used to test lipid binding of SopF according to the manufacturer’s protocol. Briefly, PIP Strips and PIP Arrays were blocked in PBS/0.1% (v/v) Tween-20 (PBST) containing 1% (w/v) skim milk powder (PBST-milk) for 1 h at room temperature. Purified recombinant GST fusion protein was incubated with PIP Strips and PIP Arrays at a concentration of 1 μg/ml in PBST containing 3% (w/v) bovine serum albumin (PBST-BSA) for 1 h at room temperature. PIP Strips and PIP Arrays were washed 3 times with PBST, then probed with rabbit anti-GST antibody (1:2,000; Cell Signaling) in PBST-BSA for 1 h at room temperature. PIP Strips and PIP Arrays were washed 3 times with PBST, then incubated with anti-rabbit HRP-conjugated secondary antibodies (1:3,000; Perkin Elmer) in PBST-milk at room temperature for 1 h. Chemiluminescence signal was detected using Clarity Western ECL Substrate (Bio-Rad) and an Amersham Imager 600 machine.

To test the binding properties of MtDef5 and its γ-core motif variants, a protein-lipid overlay experiment was performed using PIP Strips^TM (2 × 6 cm nitrocellulose membranes) that are spotted with 100 pmol of various biologically active lipids, respectively (Echelon Biosciences, Salt Lake City, UT). To determine relative degree of the binding of MtDef5, PIP Array (P-6001, Echelon Biosciences, UT) pre-spotted with the concentration gradient of various phosphoinositides was used. The MtDef5-phospholipid interactions were detected using the affinity-purified MtDef5-derived peptide (CQKRSTTWSGP) polyclonal antibody (GenScript, Piscataway, NJ) and HRP-conjugated Goat Anti-Rabbit IgG secondary antibody following the manufacturer’s protocol with minor modifications as described previously^{14 (link)}. The binding of the peptide polyclonal antibody to MtDef5 was confirmed by Western blot analysis (Supplemental Figure S7). The specificity of the binding of polyclonal antibody used here to MtDef5 was confirmed by Western blot analysis (Figure S8).

Lipid arrays (Echelon Biosciences, Salt Lake City, UT, USA) used in this work are PIP Strip (P6001), Membrane Lipid Strip (P6002), Membrane Lipid Array (P6003), PIP Array (P6100), and Sphingo Array (S6001). After saturation (3% bovine serum albumin and 0.1% Tween 20 in TBS buffer), membranes were incubated overnight with 5 mL of the protein of interest dissolved in the same buffer. After 4 washes with 0.1% Tween 20 in TBS buffer, the lipid-bound protein was detected upon 1-h incubation with the relevant HRP-labelled anti-tag antibody, followed by a further 4 washes. Detection antibodies were visualised and quantified using Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Waltham, MA, USA) and a ChemiDoc imager (Bio-Rad, Hercules, CA, USA). The software automatically checks saturation and auto-scaled images to optimise signal/noise.