Polyvinylidene difluoride membrane

SDS- polyacrylamide gel electrophoresis (PAGE) was used to separate protein samples according to their electrophoretic mobility [56 (link)]. The separated proteins were stained with Coomassie Brilliant Blue R250 or electro-blotted on a polyvinylidene difluoride membrane (Roth) with a semi-dry blotter (Bio-Rad) [57 (link)]. For Western blot analyses, antibodies directed against the His-tag (monoclonal, horseradish peroxidase (HRP)-conjugated, Novagen, 1:1000), the cyt. b₆ N-terminus (SKVYDWFEER, polyclonal, rabbit, Gramsch Laboratories, Munich, Germany, 1:10000) or the cyt. b₆ C-terminus (EIRKQGISGPL, polyclonal, rabbit, Gramsch Laboratories, Munich, Germany, 1:10000) were used. In case of the latter ones, anti-rabbit antiserum (HRP-conjugated (1:10000), Sigma-Aldrich) was used as a secondary antibody. If HRP-conjugated antibodies were used, membranes were developed using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Inc.), and chemiluminescence was detected using a Stella 3200 (Raytest).

E. faecalis lysates were subjected to SDS-PAGE and analyzed by Western blot. Separated proteins were transferred onto a polyvinylidene difluoride membrane (Roth, Karlsruhe, Germany) and blocked at 20–22°C for 2 hours in TBST (0.05% Tween 20 in TBS), containing 3% skim milk (AppliChem, Darmstadt, Germany), then incubated for 18 hours at 4°C with the polyclonal rabbit antibodies raised either against enterococcal lipoteichoic acid (LTA) [67 (link)] or against whole bacteria (E. faecalis strain 12107) [68 (link)]. After washing in TBST the membrane was incubated at 20–22°C for 1 hour with goat anti-rabbit immunoglobulin G (IgG) (whole molecule) alkaline phosphatase conjugate (Sigma Aldrich, Taufkirchen, Germany) and then washed again. Binding of the antibodies conjugated to alkaline phosphatase was detected with nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3'-indolyphosphate (BCIP) (both Sigma Aldrich, Taufkirchen, Germany).

Cells were lysed in RIPA buffer (Merck) supplemented with 2 mM NaF, 2 mM orthovanadate, 1 mM DTT, and 1× protease inhibitor cocktail (Sigma-Aldrich) or by boiling in lysis buffer (300 mM Tris–HCl pH 6.8, 6% SDS, and 25% glycerol) for 5 min at 95°C. Lysates were sonicated and protein concentrations were measured using Bio-Rad Bradford solution (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal amount of protein was resolved by SDS–PAGE (8% or 10% gels) and transferred to nitrocellulose membrane (Amersham Protran; Sigma-Aldrich) or polyvinylidene difluoride membrane (Carl Roth GmbH) by wet-transfer. Membranes were blocked in 5% milk or 5% BSA diluted in 0.05% Tween-20 in PBS (PBS-T) for 1 h at room temperature followed by incubation with primary antibodies (Table S4) diluted in blocking solution overnight at 4°C and incubation with horseradish peroxidase conjugated secondary antibodies (Jackson ImmunoResearch Labs) diluted 1:10,000 in 5% milk in PBS-T for 1 h at room temperature. Protein was detected by chemiluminescence using a Fusion F67 chemiluminescence reader (Vilber Lourmat). The source of antibodies used is given in Table S4.

Table S4 Primary antibodies for immunoblotting.

To investigate the expression of the novel vIL-8 splice variant and vIL-8 secretion, Western blot analyses were performed as described previously [20 (link)]. Briefly, CECs were infected with 10,000 plaque-forming units (pfu) of the indicated viruses. Cells or supernatants were harvested at 5 days post infection (dpi). Samples were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Carl Roth; Karlsruhe, Germany) using the Biometra semi-dry blotting system (Biometra; Göttingen, Germany). Subsequently, the membranes were blocked with 5% milk in phosphate-buffered saline (PBS) and incubated overnight at 4 °C with a mouse monoclonal α-FLAG tag antibody (1:1000; ABM; Richmond, Canada), the rabbit polyclonal anti-vIL-8 antibody [8 (link)], or the mouse monoclonal anti-gC antibody [9 (link),21 (link)], respectively. After three washes with PBST (PBS containing 0.05% Tween 20), the membranes were incubated for one hour at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit antibodies (1:10,000; Cell Signaling; Danvers, MA, USA). Finally, membranes were visualized using enhanced chemiluminescence (ECL) plus substrate (Thermo Fisher Scientific; Waltham, MA, USA), and protein signals were visualized with the Chemi-Smart 5100 detection system (Peqlab; Erlangen, Germany).

Cells were lysed in RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X 100, 0.5 sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 20 mM β-glycerophosphate plus Complete protease inhibitors (Roche, Basel, Switzerland). For lysates from 3D cultures, cells were cultured on pure matrigel without collagen. Spheroids were isolated after 4 days by dissolving the matrigel in ice cold Cell Recovery Solution (BD). After centrifugation the spheroids were lysed in RIPA buffer. Lysates were clarified by centrifugation. Equal amounts of protein were separated by SDS–PAGE (NuPAGE^® Novex Bis-Tris Gel; Invitrogen) and transferred to nitrocellulose membrane (iBlot^®Gel Transfer Stacks; Invitrogen). Alternatively lysates were loaded on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Roth). Membranes were blocked with 0.5% blocking reagent (Roche) in PBS containing 0.1% Tween-20 and incubated with primary antibodies, followed by HRP-conjugated secondary antibodies. Visualization was done with ECL detection system (Pierce, Rockford, IL, USA). Alternatively detection was done with IR-labled secondary antibodies IRDye 800 CW goat anti-mouse IgG (Licor Biotechnology, Bad-Homburg, Germany) and IRDye 680 LT goat anti-rabbit IgG (Licor Biotechnology).

Cells were lysed in RIPA buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X-100, 0.5% sodium deoxycholate, 1 mM EDTA, 0.5 mM PMSF, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 20 mM β-glycerophosphate plus Complete protease inhibitors without EDTA (Roche)] and lysates were clarified by centrifugation (16,000 × g, 10 min). Protein concentration was determined by Bio-Rad DC protein assay. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roth, Karlsruhe, Germany). Alternatively, lysates were loaded on 4–12% NuPAGE® Novex Bis–Tris gels (Invitrogen) and transferred to nitrocellulose membranes (iBlot®Gel Transfer Stacks; Invitrogen). Membranes were blocked with 0.5% blocking reagent (Roche) in PBS containing 0.05% Tween-20 and incubated with primary antibodies, followed by HRP-labeled secondary antibodies for ECL-based (Pierce, Rockford, IL) visualization with the Amersham600 system (GE Healthcare) or the Fusion Solo (VilberLourmat). Original western blots of all cropped blots are provided as Supplementary Information.

80% confluent cells were directly lysed in 6-well plate with NP 40 buffer containing 5 mm NaF and a protease inhibitor cocktail (Roche) and homogenized by ultrasound (Bandelin Sonoplus, Berlin, Germany). In brief, following 20 min incubation on ice, samples were centrifuged at 8000 r.p.m. for 10 min. Supernatants were isolated and protein concentration measured with the NanoVue Plus Spectrophotometer (GE Healthcare). Samples were mixed with loading buffer (4 ×) and NuPAGE Sample Reducing Agent (10 ×) (Invitrogen) and boiled at 96 °C for 8 min. Equal amounts of protein lysates were loaded and separated by 10–12% SDS-NuPage gel (Invitrogen) and electrophoresis was performed in MOPS-buffer, transferred to polyvinylidene difluoride membranes (Roth). Pretreatment of membranes was performed in PBS containing 2% Magic block (containing 10% Top block (Lubio Science, Luzern, Switzerland)) for 1 h. The membranes were then hybridized with antibodies against Nrf2 (1:200), Keap1 (1:1000), xCT (1:200) and β-actin (1:3000) in 5% BSA-TBST for 10 h at 4 °C. ECL detection was conducted with horseradish peroxidase-conjugated secondary antibody incubation and ECL plus kit (GE Healthcare).