Protease inhibitor mix

A mixture of NDC80 and T_xS_y with a 3–4 fold molar excess of NDC80 per S subunit was incubated for 12–20 hr at 10°C in the presence of PMSF (1 mM) and protease inhibitor mix (Serva). The formation of T_xS_y-SPC24^SPY_y was monitored using SDS-PAGE followed by coomassie staining (samples not boiled). We either used a sortase-labeled fluorescent NDC80 complex or included GGGGK-TMR peptide and a sortase 7M mutant (Hirakawa et al., 2015 (link)) in the overnight spy-coupling reaction. In the latter case, molar ratios of approximately 20 and 0.2 compared to NDC80 were used. Reaction mixtures were applied to a Superose 6 increase 10/300 or a Superose 6 increase 5/150 column (GE Healthcare) equilibrated in 20 mM TRIS pH 8.0, 200 mM NaCl, 2% v/v glycerol, 2 mM TCEP. Size-exclusion chromatography was performed at 4°C under isocratic conditions at recommended flow rates and the relevant fractions were pooled and concentrated using 30 kDa molecular mass cut-off Amicon concentrators (Millipore), flash-frozen in liquid nitrogen, and stored at −80°C.

The native PLC was isolated from Burkitt’s lymphoma cells (Raji ATCC^® CCL-86), cultured in RPMI 1640 medium (Gibco), supplemented with 10% Fetal Calf Serum (FCS, Capricorn), 3 mM HEPES-NaOH pH 7.5 (Gibco) at 37 °C and 8% CO₂ in a shaking incubator (Eppendorf). Cells were harvested by centrifugation, snap frozen in liquid nitrogen, and stored at −80 °C until further use. Cell pellets were thawed and resuspended in 20 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 10 mM MgCl₂, protease inhibitor mix (Serva) and incubated with ICP47^SBP for 15 min at 4 °C. Membranes were mixed with 2% (w/v) glyco-diosgenin (GDN, Anatrace) by douncing and incubated for 2 h at 4 °C under agitation. Insoluble material was removed by centrifugation (45 min, 100,000 × g). ICP47^SBP-arrested PLC was bound to Streptavidin High-Capacity Agarose (Pierce) and washed extensively. The PLC was either directly eluted in 20 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.05% (w/v) GDN, 2.5 mM biotin (PLC::GDN) or reconstituted into MSP2N2 nanodiscs on the beads (PLC::MSP2N2).

MBP-Knl1^138–168-H6 and MBP-Knl1^138–168-H6 constructs (MELT1) were obtained by sub-cloning into a pGEX vector backbone in which the coding sequence for GST was replaced with that for MBP. Expression was carried out in BL21 RIL strain at 25°C and by using 1 mM IPTG for 2.5 hr to induce expression. Cell pellets were re-suspended in three pellet volumes of 50 mM HEPES-NaOH pH 7.5, 250 mM KCl, 2 mM DTE, 10% glycerol, protease inhibitor mix (Serva). Cells were lysed by sonication, and the lysates were centrifuged at 100000×g for 1 hr at 4°C. Recombinant products were isolated from the lysate by using the HisTrap (GE Healthcare) column, followed by buffer exchange using a desalting column (GE Healthcare). Purified proteins were concentrated to about 3 mg/ml and frozen in liquid nitrogen.

The proteins were extracted from the muscle, brain, testis, and ovary in RIPA lysis buffer (TCL131; HIMedia^® Laboratories GmbH, Homburg, Germany) supplemented with a protease inhibitor mix (39102.01; SERVA Electrophoresis GmbH, Heidelberg, Germany). The samples were sonicated three times (20 Hz for 20 s each), placed on ice for 30 min, and then centrifuged at 10,000× g for 30 min at 4 °C. The supernatants were collected. Forty micrograms of the protein extracts was separated into SDS-PAGE (9–15% acrylamide) and treated according to Romano et al. [41 (link)]. Then, they were incubated overnight at 4 °C with primary antibodies as follows: anti-INSL3 (1:800) and anti-α-Tubulin (1:5000, E-AB-20036, Elabscience^®, Houston, TX, USA). After the incubation, the filters were washed three times in TBST and incubated with peroxidase-conjugated secondary antibody anti-mouse IgG (1:5000, AP130P, Sigma Aldrich^®, Munich, Germany) for the mouse and anti-α-Tubulin or anti-rabbit IgG (1:3000 AP307P; Sigma-Aldrich^®, Munich, Germany) secondary antibody for the rabbit anti-INSL3 for 1 h at RT. Then, the filters were washed in TBST three times. The immunocomplexes were detected using the enhanced chemiluminescence (ECL) WB detection system. ImageJ software (version 1.53 g; NIH) was used to analyze all the bands.