Pierce micro bca assay

Protein extraction was performed on 7-day old P and ceh1 seedlings grown on a 1/2 × MS media. Tissue was frozen upon collection and grind in liquid nitrogen using protein extraction buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 2 mM EGTA, 0.01% SDS, 1 mM DTT, 10 μM/ml Protease Inhibitor Cocktails (Sigma)). The extract was subsequently centrifuged at 10,000 RPM for 15 min at 4 °C. The supernatant was collected and protein concentration was measured using Thermo Scientific Pierce Micro BCA Assay according to manufacturer instructions. Protein concentration of samples were adjusted to 2 or 3 μg/μL in 5 × Laemmli Buffer, heated at 56 °C and subsequently separated on 10% SDS-PAGE gel and transferred onto nitrocellulose membranes. Blots were probed with anti-PIN1 monoclonal antibody (1:100)^{63 (link)} primary antibody and secondary anti-mouse- Horseradish peroxidase (HRP) (KPL, catalog no. 074-1806) (1:3000) and chemiluminescent reaction was performed using and Pierce ECL Western Blotting Substrate. Working solutions of the substrates were prepared according to the manufacturers’ instructions and added to the membranes. The membranes were placed in plastic sheet protectors. Each membrane was exposed to X-ray films and developed. The uncropped scans of the blots are shown in Supplementary Figs. 1d and 6b. The anti-PIN1 monoclonal antibody was produced in Klaus Palme’s laboratory.

Cells in mock community samples were disrupted by bead-beating (6.0 m/s, 45 s) in lysing matrix B tubes (MP Biomedicals) in SDT lysis buffer [4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT] followed by heating to 95°C for 10 min. Tryptic digests of protein extracts were prepared according to the filter-aided sample preparation (FASP) protocol described by Wiśniewski et al. (2009) (link). Peptides were desalted with Sep-Pak C18 Plus Light Cartridges (Waters). Acetonitrile from the peptide elution step was exchanged for 0.1% formic acid (v/v) using a centrifugal vacuum concentrator. The desalting step was necessary to enable binding of peptides to the SCX column during sample loading for the 2D-LC methods. Peptide concentrations were determined using the Pierce Micro BCA assay (Thermo Scientific Pierce) according to manufacturer’s instructions. The peptide mixture was aliquoted and frozen at −80°C. For mass spectrometric analyses, fresh aliquots were regularly thawed and formic acid concentration increased to 0.2% (v/v). All aliquots used here were prepared at the same time from the same peptide mixture to eliminate between-sample variation.

Subcultures of E. faecalis overnight cultures were infected with VPE25 as described previously (30 (link)). Four samples of 4 mL each were taken at 0, 10, 20, and 40 minutes after VPE25 treatment and pelleted. Pelleted samples were resuspended in 300 μL of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl, 0.1 M DTT). Cells were lysed by bead-beating using a Bead Ruptor Elite (OMNI) with Matrix Z beads (MP Biomedicals) for two cycles of 45 s at 6 m/s. Samples were incubated at 95°C for 10 min. Tryptic digests of protein extracts were prepared following the filter-aided sample preparation (FASP) protocol described previously, with minor modifications as described in Kleiner et al. (63 (link), 64 (link)). Lysate was not cleared by centrifugation after boiling the sample in lysis buffer. The whole lysate was loaded onto the filter units used for the FASP procedure. Centrifugation times were reduced to 20 minutes as compared to Kleiner et al. (64 (link)). Peptide concentrations were determined with the Pierce Micro BCA assay (Thermo Scientific) using an Epoch2 microplate reader (Biotek) following the manufacturer’s instructions.