Chemostar ecl imager

Adherent cells (RAW264.7, HepG2, AML-12) were seeded into 6-well plates 24 h prior to the experiment. All cells were washed 3 times with phosphate-buffered saline (PBS) and afterwards starved in serum-free medium for 2 h (Ba/F3-gp130-hIL-6R and Ba/F3-gp130-mIL-6R) or 4 h (all other cell types). The cells were incubated with the indicated amounts of tocilizumab for 30 min and then stimulated with 10 ng/ml hIL-6 or mIL-6 (for human or murine cells, respectively) for 15 min. Subsequently, cells were harvested, lysed, and 40 μg protein were separated by SDS-PAGE and blotted onto nitrocellulose membranes. After blocking, membranes were incubated with primary antibodies at 4°C over night and, after washing, with AlexaFluor-conjugated or IRDye-conjugated secondary antibodies for 1h at room temperature in the dark. Signals were detected using the a ChemoStar ECL Imager (Intas, Göttingen, Germany) or an Odyssey Fc Imager (LI-COR Biosciences, Bad Homburg, Germany), and densitometric analysis was performed using the ImageStudio software (LI-COR Biosciences, Bad Homburg, Germany). Data are presented as ratio of the pSTAT3/STAT3 signals from three independent experiments (mean ±SD). Additionally, one representative western blot per experimental setting is shown.

Mouse brain synaptosomes were generated as detailed by Rummel et al. [58 (link)]. Briefly, functional synaptosomes were recovered from a Percoll gradient after homogenisation of cerebrum obtained from 6–10 mouse brains and various centrifugation steps, and finally diluted in physiological buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 20 mM HEPES, 10 mM glucose, 0.5% BSA, pH 7.4) with the final synaptosomal protein adjusted to a concentration of 10 mg/mL. Lysates of MNs and SiMa cells not treated with BoNTs were pooled from three independent differentiations. Totals of 10 µg protein each were additionally separated on a 12.5% gel via SDS-PAGE and transferred onto PVDF membranes (Roti^®-PVDF, pore size 0.45 µm, ROTH, Karlsruhe, Germany). Membranes were blocked with 5% milk powder in PBST (PBS, 0.05% Tween) for 1 h. Anti-SV2A, anti-SV2B, anti-SV2C, anti-SYT1 and anti-SYT2 primary antibodies (Table A1) were diluted in 5% milk powder in PBST and incubated over night at 4 °C. HRP-coupled secondary antibodies (both from Rockland, Limerick, PA, USA, Table A2) were diluted 1:20,000 and incubated for 4 h at room temperature. Bands were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) with the ChemoStar ECL Imager (Intas, Göttingen, Germany).

Cells were harvested and lysed with RIPA buffer (Cell signaling, 9806) in ice for 30 min and centrifuged at 12000× g for 10 min. Supernatant was collected and protein concentration was measured using BCA method (Thermo Fisher Scientific). Protein samples were boiled in 1x NuPAGE LDS sample buffer (Invitrogen) at 70℃ for 10 min. Twenty microgram protein samples were subjected to 7.5% - 15% gradient SDS-PAGE gel and transferred to PVDF membrane (MACHEREY-NAGEL, Germany). The membranes were blocked in 1x Roti-Block (Carlroth, Germany) at room temperature for 1 h and were then probed with specific primary antibodies overnight at 4°C. Blots were incubated with HRP-conjugated secondary antibody (Invitrogen, 31430 and 31460) for 1 h at room temperature. Bands were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and detected using the ChemoStar ECL Imager (Intas Science Imaging, Germany).

Protein extraction was done from homogenized tissues, re-suspended in 2 × volumes of high salt buffer (HSB) (Tillmann et al. 2015 ). Protein extraction from protoplasts was done after resuspension of cells to 60 μl HSB (Hahn et al. 2011 (link)). The samples were subsequently centrifuged for 5 min at 14,000 rpm and 4 °C. The supernatant was supplemented with 4 × SDS loading buffer and boiled at 95 °C for 5 min. Extracts were separated on 10 or 12% SDS–polyacrylamide gels. For immunoblot analysis, proteins were transferred to a nitrocellulose membrane (Protran nitrocellulose transfer membrane; Whatman) and protein signals were detected using chemiluminescence following the manufacturer’s protocol (Western Lightning Plus ECL solutions; Perkin-Elmer). The antibodies used were GFP (Roche), hemagglutinin tag (HA; Covance), and actin (Sigma-Aldrich). Antibodies used for detection of HsfA1a have been previously described (Lyck et al. 1997 (link)). Immunosignals were visualized with horseradish peroxidase (HRP)–coupled secondary antibodies (Sigma-Aldrich) and recorded in a ChemoStar ECL imager (INTAS, Göttingen, Germany).

Twenty microgram protein samples were electrophoresed on a 7.5–15% gradient SDS-PAGE gel (Tris-Glycine, self-made) and transferred to PVDF membrane (MACHEREY-NAGEL, Dueren, Germany) by semi-dry electroblotting (Bio-Rad, Singapore). The membranes were blocked for 1 h in 1× Roti-Block (Carl Roth, Karlsruhe, Germany) at RT and then incubated with specific primary antibodies at 4 °C overnight. Proteins were detected after incubation with HRP-conjugated secondary antibody (Invitrogen, 31430 and 31460) for 1 h at RT and visualized with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and detected by ChemoStar ECL Imager (Intas Science Imaging, Göttingen, Germany).