Spin column

L. lactis NCDO 2118 and L. lactis IL1403 were pre-inoculated in M17 medium (Difco, New Jersey, USA) and incubated at 30°C for 16 h. The precultures were then inoculated (1:100) in fresh M17 medium supplemented with 0.5% (w/v) glucose (M17Glc) at 30°C until reaching an OD₆₀₀ = 0.8 (three independent experiments). The cultures were then centrifuged for 20 min at 2,700 x g. The supernatants were filtered using 0.22-μm filters, 30% (w/v) ammonium sulfate was added to the samples, and the pH of the mixtures was adjusted to 4.0. Next, 20 mL of N-butanol was added to each sample. The samples were centrifuged for 10 min at 1,350 x g and 4°C. The interfacial precipitate was collected and resuspended in 1 mL of 20 mM Tris-HCl pH 7.2 [102 (link)]. To perform label-free proteomic analysis, the protein extract was concentrated using a spin column with a 10 kDa threshold (Millipore, Billerica, MA, USA). The protein was denatured (0.1% RapiGEST SF at 60°C for 15 min) (Waters, Milford, CA, USA), reduced (10 mM DTT), alkylated (10 mM iodoacetamide) and enzymatically digested with trypsin (Promega, Sequencing Grade Modified Trypsin, Madison, WI, USA).

For proteomic analysis, three independent control and recovered colonies from the three individual frozen stocks were grown in CDM to an OD600 = 0.8. The cultures were then centrifuged for 20 min at 2,700 × g. The supernatants were filtered using 0.22-μm filters, 30% (w/v) ammonium sulfate was added to the samples, and the pH of the mixtures was adjusted to 4.0. Next, 20 mL/L N-butanol was added to each sample. The samples were centrifuged for 10 min at 1,350 × g and 4°C. The interfacial precipitate was collected and resuspended in 1 mL of 20 mM Tris-HCl, pH 7.2 (Paule et al., 2004 (link)). Proteins were quantified using the Bradford assay. For label-free proteomic analysis, the protein extract was concentrated using a spin column with a 10 kDa threshold (Millipore, Billerica, MA, USA). The protein was denatured (0.1% RapiGEST SF at 60°C for 15 min) (Waters, Milford, CA, USA), reduced (10 mM DTT), alkylated (10 mM iodoacetamide) and enzymatically digested with trypsin (Promega, Sequencing Grade Modified Trypsin, Madison, WI, USA). Glycogen phosphorylase (Waters Corporation, SwissProt P00489) was added to the digests to a final concentration of 20 fmol/μl as an internal standard for normalization prior to each replicate injection. The digestion process was stopped by adding 10 μL of 5% TFA (Fluka, Buchs, Germany) (Silva et al., 2006 (link)).

We adapted our PEG-switch assay that replaces palmitate with a 5-kDa methoxy PEG (21 (link)) by using a refinement that improves the reaction efficiency by PEGylating separately from the thioester cleavage step (53 (link), 54 ). Briefly, free protein thiols were alkylated with 100 mM maleimide (in the presence of 2.5% sodium dodecyl sulfate (SDS), 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5) for 4 h at 40 ˚C. Excess maleimide was removed using acetone precipitation, followed by extensive washing of the protein pellet with 70% acetone. Proteins were resolubilized (1% SDS, 100 mM HEPES, and 1 mM EDTA, pH 7.5), and thioester bonds cleaved using 200 mM neutral hydroxylamine for 1 h at 37 ˚C. Hydroxylamine was removed by desalting (Zeba spin column), and free cysteines were PEGylated with 2 mM 5K-PEG maleimide (Sigma) for 1 h at 37 ˚C.
Palmitoylated proteins were purified from whole-cell lysates using acyl-RAC. Free thiols were alkylated with methyl methanethiosulfonate and palmitoylated proteins captured using thiopropyl Sepharose in the presence of neutral hydroxylamine. (44 (link))

Affinity purification coupled with mass spectrometry (AP-MS) experiments were performed as previously described^{14 (link)}. For protein purification, HEK293 cell lines stably expressing IRF5-FLAG divided into 2 groups. Each group of cells was expanded and cultured in five 15-cm dishes. Then, one group was treated with 1 μg/ml R848. After 16 hr, the cells were collected and lysed in 10 ml of TAP buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 100 mM NaCl, 0.5% Nonidet P40, 10% glycerol, phosphatase inhibitors and protease inhibitors)^{28 (link)}. Cell lysates were precleared with 50 μl of protein A/G resin before the addition of 20 μl of anti-FLAG resin and incubation for 16 hr at 4 °C on a rotator. The resin was washed 3 times and transferred to a spin column (Sigma) with 40 μl of 3X FLAG peptide for 1 hr at 4 °C on a rotator. The purified complexes were loaded onto a 4–12% NuPAGE gel (Invitrogen, Catalog #NP0323BOX). The gels were stained with a SilverQuest staining kit (Invitrogen, Catalog #LC6070), and lanes were excised for mass spectrometry analysis by the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA).

Mass spectrometry experiments were performed as previously described [38 (link),39 ,59 (link)]. For protein purification HEK293 stable cell lines expressing FLAG-PB1 were collected from five 15 cm dishes in 10 ml TAP buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 100 mM NaCl, 0.5% Nonidet P40, 10% glycerol, phosphatase inhibitors and protease inhibitors) [32 (link)]. Cell lysates were precleared with 50 μl protein A/G resin before addition of 20 μl anti-FLAG resin (Sigma) and 16 hr incubation at 4°C on a rotator. The resin was 3X washed and transferred to a spin column (Sigma) with 40 μl 3X FLAG peptide (Sigma) for 1 hr at 4°C on a rotator. Purified complexes were loaded onto a 4–15% NuPAGE gel (Invitrogen). Gels were stained using the SilverQuest staining kit (Invitrogen) and lanes were excised for mass spectrometry analysis by the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA).

HEK293 cell lysate (100 μg) was labeled with Cy5 (GE Healthcare Bio-Sciences Corp., Marlborough, MA, USA) and free dyes were abandoned by Spin column (Sigma Aldrich, Saint Louis, MO, USA). To make 5-HT2c receptor microarray, a glass slide covered with super epoxy group (Arrayit Corporation, Sunnyvale, CA, USA) was used. The antibody microarray using the glass chip was prepared by immobilizing 42 antibodies against cell cycle proteins. Cy5-labeled tryptamine and GM were applied to the Protein Chip and incubated for 1 h at 37 ◦C. The Protein Chip was then washed with PBST and DW and dried under a stream of N2 gas. GM was dissolved in ethanol and adulterated to the desired concentration using PBS. The GM concentrations ranged from 1000 µM to 15.625 µg/mL. Tryptamine alone was used as the negative control. GM was used to estimate its calmative effect using a 5-HT2c receptor binding assay. The chips were scanned using a GenePix 4100 A microarray scanner (Axon Instruments, Union City, CA, USA). The internally normalized proportion of all spots was evaluated.

Hemolymph for the bacterial growth inhibition assay and proteomic analysis (below) was collected by cutting the first two leg pairs of cockroaches that had been pre-chilled on ice for 15 min. They were then placed head-down into a spin-column (Sigma-Aldrich) in a 1.5 ml tube containing approximately 10 μg propylthiouracil (to inhibit phenol-oxidase activity), and centrifuged at 500 g for up to 5 min or until at least 10 μl of hemolymph were collected. The entire operation was carried out in a pre-cooled centrifuge at 2 °C.