Precision plus dual xtra protein standards

To determine the oligomeric status of MtDef5 and its γ-core motif variants, protein cross-linking experiments were performed in the presence or absence of PIP, PA and PI cross-linked with bis[sulfosuccinimidyl] suberate (BS^{3 (link)}) substrate^{15 (link)}. Briefly, MtDef5 and its γ-core motif variants, prepared in 1X PBS buffer, at 1.5 mg/ml (5 µL) were incubated with 2.73 mM PI3P, PI4P or PI5P (1.5 µL) at room temperature for 30 min. The cross-linking reaction was initiated by addition of freshly dissolved water soluble 12.5 mM BS^{3 (link)} (0.5 μL) and incubated for 30 min at room temperature. After crosslinking, samples were reduced with 100 mM dithiothreitol (DTT) and separated on a 4–20% (Bio-Rad, TGX Stain-Free™ precast gels) SDS-PAGE. The oligomerization pattern was visualized using a Bio-Rad ChemiDoc XRS + system. For SDS-PAGE, Precision Plus Dual Xtra Protein Standards (2–250 kD) from Bio-Rad (Hercules, CA) were used.

Preferential binding of the MtDef5 and its γ-core motif variants to PIP was further validated by a liposome binding assay. PolyPIPosome binding assays were performed in 200 µL of binding buffer (20 mM HEPES pH 7.4, 120 mM NaCl, 1 mM EGTA, 1 mM MgCl₂, 1 mg/mL BSA, 0.2 mM CaCl₂, 5 mM KCl) containing 10 µL each of control PolyPIPosome, PI PolyPIPosome, PI3P PolyPIPosome, PI4P PolyPIPosomes and PI5P PolyPIPosome (Echelon Biosciences). Subsequently, a total of 1.25 µg each of MtDef5 and its γ-core motif variants was added and incubated for 1 h at room temperature. Liposomes were collected by centrifugation at 16, 000 × g for 20 min, and supernatant saved and the pellet was washed three times in 1 mL of binding buffer. Following three washes, pellet was resuspended in 40 µL of 2x Laemmli sample buffer and an aliquot of 20 µL of the supernatant was mixed with 20 µL of sample buffer. Both samples were reduced with 100 mM dithiothreitol (DTT) and separated on a 4–20% (Bio-Rad, TGX Stain-Free™ precast gels) SDS-PAGE gel. The proteins were visualized using a Bio-Rad ChemiDoc XRS + system. Stain-free gels contain a special Trihalo compound, which, when activated, reacts with tryptophan residues in the protein sample to emit a fluorescence signal. For SDS-PAGE, Precision Plus Dual Xtra Protein Standards (2–250 kD) from Bio-Rad (Hercules, CA) were used.

Protein samples were mixed with 4X Protein Loading Buffer (Li-COR 926–31097) and 1:20 DTT (1M DTT) and denatured at 95°C for 5 minutes. Proteins were separated by SDS-PAGE on NOVEX-NuPAGE 4–12% BIS-TRIS gels with MES Running Buffer and Precision Plus Dual Xtra Protein Standards (Bio-Rad161-0377) were used to estimate the molecular weight of proteins. For Western blots, the SDS-PAGE separated proteins were transferred to nitrocellulose membranes. Non-specific binding to the membrane was blocked with Odyssey PBS Blocking Buffer (Fisher Scientific NC9877369) and probed with the antibodies listed below. Blots were imaged on an Odyssey CLx and analyzed using ImageStudio2.1 (Li-COR). The following primary antibodies were used: monoclonal mouse anti-FLAG M2 (Millipore Sigma F3165), monoclonal mouse anti-strep (StrepMab) Classic iba LifeSciences 2-1507-001), custom generated Rabbit anti-Cbp1 (Rb83), custom generated Rabbit anti-RYP1, mouse anti-LAMP1 (cell signaling D401S), mouse anti-alpha-tubulin (Novus biological DM1A), Rabbit anti-calnexin (abcam 22595), mouse anti-His (ABclonal AE003).