Phenylmethylsulfonyl fluoride pmsf

Ampicillin was obtained from Wako Pure Chemical Industries Ltd (Osaka, Japan). Anhydrotetracycline and Strep-Tactin-Sepharose were purchased from IBA. Triton X-100, ethylenediaminetetraacetic acid (EDTA), urea, l-arginine hydrochloride, l-cystine dihydrochloride, l-cysteine, bovine serum albumin (BSA), Tween 20 and 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) were obtained from Wako Pure Chemical Industries Ltd. Desthiobiotin and Orlistat were purchased from Sigma-Aldrich (Saint Louis, MO). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Rabbit anti-HPL polyclonal IgGs and anti-Strep-tag II (ST II) monoclonal IgG were obtained from Abcam (Cambridge, UK) and IBA GmbH (Gottingen, Germany), respectively. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and anti-mouse IgG antibodies were from Kirkegaard and Perry Laboratories (Washington, DC, USA). Tributyrin was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). Porcine colipase was from AbD Serotec (Kidlington, UK). Gel-filtraion FPLC columns, HiPrep 16/60 Sephacryl S-200 HR were purchased from GE Healthcare.

pCDNA-GCK-HA and ubiquitin-Myc (Fig. 5c, d) expression vectors were transfected to HEK293FT cells using Lipofectamine^® 2000 (Thermo Fisher). Transfection efficiency was ~90%. The cells were cultivated in DMEM and lysed in buffer containing 20 mmol/l HEPES-NaOH (pH 7.9), 1 mmol/l MgCl₂, 0.2 mmol/l CaCl₂, 100 mmol/l KCl, 0.2 mmol/l EDTA, 10% Glycerol, 0.1% Nonidet P-40, 1 mmol/l Dithiothreitol, 0.2 mmol/l Phenylmethylsulfonyl fluoride (PMSF, Nacalai Tesque, Kyoto, Japan), 3% n-Octyl-β-D-glucoside (DOJINDO, Tokyo, Japan). The lysate was mixed with arginine/glucose-immobilized magnetic nanobeads (Fig. 1e, f), or directly analyzed by SDS-PAGE and Western blotting with HA or GCK antibody.

Western blotting was performed as described previously^{53 (link),54 (link)}. Briefly, the cells were lysed in sodium dodecyl sulfate (SDS) sample buffer containing protease inhibitors [10 μg/mL aprotinin (Fujifilm Wako Pure Chemicals), 4 μg/mL pepstatin A (Peptide Institute, Inc, Osaka, Japan), 10 μg/mL leupeptin (Nacalai Tesque), 2.5 mM EGTA- KOH (Sigma), 1 mM phenylmethylsulfonyl fluoride (PMSF, Nacalai Tesque)]. The whole cell lysates were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto polyvinylidene difluoride membranes (PVDF; Pall Corporation, Port Washington, NY, USA). Blocking to minimize non-specific interactions was done with Blocking One (Nacalai Tesque) or 5% BSA in Tween 20-containing Tris-buffered saline (TTBS) [20 mM Tris–HCl (pH 7.5), 137 mM NaCl, and 0.1% Tween 20] at room temperature for 30 min. Later, the membranes were incubated with the antibodies, which were diluted with TTBS containing 5% Blocking One or 3% BSA. Clarity (Bio-Rad) was used as the chemiluminescence substrate. A ChemiDoc XRSplus image analyzer (Bio-Rad) was used for the chemiluminescence detection and band intensity analysis.