Mass spectrometry grade

Proteins in excised gel spots were digested with trypsin (Mass Spectrometry Grade, Promega, Madison, WI, USA) overnight at 37 °C. Peptides were extracted with 90% acetonitrile/2.5% trifluoroacetic acid and peptide masses were measured by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) analysis (Ultraflex III MALDI-TOF/TOF, Bruker Daltonics, Billerica, MA, USA). The scanning UV wavelength was set at 355 nm and the mass scan range was 700 to 3200 Da.
MASCOT DAEMON (version 2.3.02, Matrix Science, London, UK) was used to extract the MS and MS/MS data via BioTools 3.0 software (Bruker Daltonics, Billerica, MA, USA). Database searches were performed in Mascot against the UniProt database (species: Microcystis aeruginosa, 79,051 sequences, 21,123,649 residues). The searches were performed with peptide mass tolerance of 50 ppm and fragment mass tolerance of 0.6 Da. One missing cleavage was allowed. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation was set as a variable modification. Only significance thresholds defined by the Mascot probability analysis (p < 0.05) were accepted.

Anti-GFP mAb-agarose (code. D153-8) and anti-GFP (Code. no. 598) were obtained from MBL (Nagoya, Japan). Mouse monoclonal anti-Coronin1C (Cat. no. sc-376919) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH (Cat. no. 2118S), Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 555 goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Rab44 antibody was raised in rabbits using a recombinant protein and prepared as previously described [34 (link)]. Recombinant RANKL was prepared as described previously [35 (link)]. M-CSF was purchased from Kyowa Hakko Kogyo (Tokyo, Japan). 4′,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 488 Phalloidin were from Thermo Fisher (Waltham, MA, USA). Alkaline phosphatase (ALP) (Cat. no. 10713023001), and guanosine 5′-triphosphate sodium salt hydrate (GTP) (Cat. no. G8877), the protease inhibitor cocktail, Duolink in situ PLA probe anti-mouse MINUS, anti-rabbit PLUS, detection reagent red, and wash buffers A and B were from Sigma-Aldrich (Tokyo, Japan). MCP-1 was obtained from FUJIFILM Wako (Osaka, Japan). Trypsin Gold and Mass Spectrometry Grade (Cat. no. V5280) were purchased from Promega (Tokyo, Japan). Transwell 24-well plates were obtained from Corning, Inc. (Corning, NY, USA). SpongeCol^® (collagen sponge) was purchased from Advanced BioMatrix, (Carlsbad, CA, USA).