Centrifugal filter unit

VHL^54–213•EloB/C complex was expressed by co-transforming BL21 (DE3) E. coli with XLB250 and XLB192 and inducing overnight at 16°C^{50 (link),51 (link)}. It was then purified on glutathione resin followed by on-column digestion with thrombin overnight at 4 °C. Protein released from the glutathione resin to the supernatant was concentrated via a centrifugal filter unit (10 kD cutoff, MilliporeSigma) and purified through a S75 size exclusion column (Cytiva) on the ÄKTA system. Purified VHL•EloB/C were aliquoted and stored in Storage Buffer at -80 °C.

Culture supernatants, collected to evaluate HSP expression levels, were concentrated in accord with our previously described protocol 17 (link). Briefly, same numbers of HNECs were seeded in 6-well plates and heat shock was induced. Entire amounts of culture supernatants were collected and concentrated (Centrifugal Filter Unit; MilliporeSigma, Burlington, MA, USA). Concentrated samples were then mixed (x 5) with sample buffer, and western blot was performed.

Gαi proteins were exchanged in CD buffer (10 mM K₂HPO₄, 500 μM MgSO₄, 500 μM tris(2-carboxyethyl)phosphine (TCEP)) using an Amicon centrifugal filter unit (MilliporeSigma, #UFC901024) at 3800 × g, diluted to 15 μM (pH 6.0, 6.4, 6.8, 7.2, or 7.6) and centrifuged (14,000 rpm) for 10 mins at 4 °C. Temperature dependent CD experiments were performed on a Jasco J815CD spectrometer using 15 μM WT or Gαi variants protein in a 1 mm path-length quartz cuvette (Hellma Analytics). Thermal melts were obtained at 222 nm, over a temperature range of 20–95°C, using a temperature increment of 1 C/min. The CD spectral scans were collected for Gαi proteins at different fixed temperatures (e.g. 45°C, 55°C and 65°C) by taking CD measurements every 1 nm from 200–250 nm. Secondary structure was evaluated using the online server BeStSel (Beta Structure Selection)^{9 (link)}.

Chang cells were lysed using Pro-Prep protein extraction solution (Intron, Seongnam, Korea) that included a mixture of protease inhibitors (Sigma-Aldrich). The protein concentration was determined by using the bicinchoninic acid (BCA) assay (Pierce Thermo Scientific, Rockford, IL, USA). Protein samples were loaded on a 12% SDS-PAGE gel and transferred to nitrocellulose membranes. Western blot analysis was performed using primary Abs against HMGB1 (Abcam, Cambridge, UK), β-actin (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), LDH (Santa Cruz Biotechnology Inc), and α-tubulin (Santa Cruz Biotechnology Inc), and HRP-labeled goat anti-rabbit or goat anti-mouse secondary Abs (Jackson Immuno Research Labs Inc, West Grove, PA, USA). ECL was used to reveal the signals (Thermo Scientific, Rockford, IL, USA), and band intensities were measured using the ImageJ program (NIH, Bethesda, MD, USA). To compare the HMGB1 secretion levels, cells were seeded into 60-mm plates and maintained in OPTI-MEM (Gibco, Invitrogen). UV was irradiated when the cells were semi-confluent, after which they were incubated for further 8 h. Cell culture supernatants were then collected and concentrated using a centrifugal filter unit (Millipore, Billerica, MA, USA) followed by western blot analysis using an anti-HMGB1 Ab.

Cell media was collected after four hours of treatment and centrifuged to clear it of any cellular debris. The media was then centrifuged through a Millipore Centrifugal Filter Unit (Millipore, Billerica, MA) to concentrate the protein to 200 ul. Secreted FGF19 protein levels (pg/mL) was measured by using an Elisa kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) with sample, controls, and standards assayed in duplicate, as we have previously described [15] (link). This immunoassay employed the quantitative sandwich enzyme technique which is based on a monoclonal antibody specific to the human FGF19. The range of detection is 31–554 (pg/mL) with a standard deviation of 125 pg/mL. The coefficient of variation (CV) values of intra-assay and inter-assay precision are 4.5% and 5.5%, respectively.