Supersignal west pico plus chemiluminescent hrp substrate

For the determination of protein expression, cells either untreated or incubated for 30 min with 1 and 100 nM dDAVP were lysed in LDS sample buffer (Thermo Fisher Scientific) and Western Blot analysis was performed as described [13 (link)]. Briefly, 20 μg of proteins were separated on Bolt™ 4–12% Bis-Tris mini protein gel (Thermo Fisher Scientific) and electrophoretically transferred to PVDF membranes (Immobilione-P membrane, Merck). Membranes were incubated for 1 h at RT in TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl; 0.1% Tween) containing 5% non-fat dried milk, then incubated overnight at 4 °C in TBST added with 5% BSA and anti-AVPR1A (Thermo Fisher Scientific), anti-AVPR2 (MyBiosource, distributed by Aurogene S.r.l, Roma, Italy) anti-phospho-eNOS (Ser1177) (Cell Signaling Technology, distributed by Euroclone, Pero, Italy) purified rabbit polyclonal antibodies (1:2000). Actin, detected with a monoclonal antibody (1:2000; Merck, Milano, Italy), was employed as internal standard. Immunoreactivity was visualized with SuperSignal™ West Pico Plus Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with iBright Analysis Software.

Protein expression was determined in cell lysates in LDS sample buffer (Thermo Fisher Scientific, Monza, Italy) and Western blot analysis was performed as described [29 (link)]. A total of 20 µg of proteins was separated on Bolt™ 4-12% Bis-Tris mini protein gel (Thermo Fisher Scientific) and electrophoretically transferred to PVDF membranes (Immobilione-P membrane, Merck). Membranes were incubated for 1 h at RT in blocking solution (TBST: 50 mM Tris-HCl pH 7.5, 150 mM NaCl; 0.1% Tween containing 5% non-fat dried milk) then incubated overnight at 4 °C in TBST added with 5% bovine serum albumin (BSA) and anti-MDR1 (E1Y7B, #13342), anti-MRP1 (D7O8N, #14685), anti-MRP2 (D9F9E, #12559), or anti-BCRP (D5V2K, #42078) rabbit polyclonal antibody (1:1000; Cell Signaling Technology, Euroclone, Milano, Italy). Vinculin, visualized by a mouse monoclonal antibody (1:2000; SAB4200080, Merck), was employed as internal loading control. Membranes were then incubated for 1 h in blocking solution containing HRP-conjugated secondary antibodies (anti-rabbit #7074 or anti-mouse (#7076) IgG, (1:10,000, Cell Signaling Technology). Immunoreactivity was detected by employing SuperSignal™ West Pico Plus Chemiluminescent HRP Substrate; and blot images, taken with iBright FL1500 Imaging System, were analyzed with iBright Analysis Software (Thermo Fisher Scientific).

Vero cells in six-well plates were infected with various rMVs at an MOI of 0.1. At 36–48 h post-infection, infected cells were lysed in RIPA lysis buffer (Thermo Fisher). Samples were briefly centrifuged and subjected to 4–12% gradient NUPAGE-PAGE gel (Invitrogen). After transfer to a nitrocellulose membrane (GE Healthcare), the membrane was subsequently probed with a rabbit polyclonal anti-SARS-CoV S antibody recognizing the conserved 1124 aa–1140 aa epitope (ABIN199984, Antibody Online, 1:2000 dilution) followed by a horse-radish peroxidase (HRP)-conjugated swine anti-rabbit IgG antibody (P0399, Dako, 1:3000 dilution). Bands were visualized using SuperSignal West Pico Plus chemiluminescent HRP substrate (Thermo Fisher). For loading controls, membranes were stripped with 5% NaOH for 5 min and re-probed with a mouse monoclonal anti-MV-N antibody (ab9397, Abcam, 1:20,000 dilution) followed by an HRP-conjugated anti-mouse IgG (NA931V, GE Healthcare, 1:10,000 dilution).

For the determination of protein expression, cells were lysed in RIPA buffer supplemented with a cocktail of protease inhibitors (cOmplete™, Mini, EDTA-free, Roche, Basel, Switzerland). Western Blot analysis was performed, as previously described [27 (link)]. Briefly, 20 µg of proteins was separated on SDS-PAGE (Bolt™ 4–12% Bis-Tris mini protein gel, Thermo Fisher Scientific) and electrophoretically transferred to PVDF membranes (Immobilon-P membrane, Merck). Membranes were incubated for 1 h at RT in Tris-buffered saline solution (TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl) containing 5% non-fat dried milk, then incubated overnight at 4 °C in TBST (TBS + 0.5% Tween) supplemented with 5% BSA and anti-ICAM-1, anti-VCAM-1, or anti-tissue factor (TF) purified rabbit polyclonal antibodies or anti-CD31/PECAM mouse monoclonal antibody (1:2000, Cell Signaling Technology, Beverly, MA, USA). Anti-vinculin (Merck) or anti-β-actin (Cell Signaling Technology) mouse monoclonal antibodies (1:2000) were employed as the internal standard. Immunoreactivity was visualized with SuperSignal™ West Pico PLUS Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with the iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with the iBright Analysis Software.

Proteins were extracted from male ileum samples and homogenized in TRI Reagent® lysis buffer (Sigma-Aldrich) according to the manufacturer’s instructions. Twenty micrograms of total protein lysate was separated by SDS-PAGE and electroblotted on nitrocellulose membranes using an Amersham Semi Dry Transfer Unit at 0.8 mA/cm² (Amersham Pharmacia Biotech). Membranes were blocked for 1 h in StartingBlock™ T20 (TBS) with 5% milk blocking buffer (Thermo Fisher Scientific) at room temperature. Then membranes were incubated overnight at 4 °C with 1:500 of rabbit anti-ZO-1 (Thermo Fisher, 61–7300) and 1 h at room temperature with 1:10,000 of mouse anti- β-actin (Sigma, A1978). Immunoreactivity was visualized with SuperSignal™ West Pico Plus Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Images were taken using the iBright FL1500 Imaging System (Thermo Fisher Scientific). For relative quantification, the volume intensity of the bands was obtained using iBright software.

The analysis of protein expression was performed as described previously [22 (link)] on cell lysates obtained with LDS sample buffer (Thermo Fisher Scientific). An amount of 20 µg of proteins were separated on Bolt™ 4–12% Bis-Tris mini protein gel (Thermo Fisher Scientific) and transferred to PVDF membranes (Immobilon-P membrane, Merck). Membranes were incubated for 1 h at RT in blocking buffer (Tris-buffered saline solution—TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl) added with 3% non-fat dried milk and then overnight at 4 °C with primary antibodies in TBST (TBS + 0.5% Tween) containing 5% BSA. Anti-phospho-NF-κB p65 (Ser536), anti-phospho-IκBα (Ser32/36) anti-phospho-STAT1 (Tyr701), anti-phospho-STAT3 (Tyr705) and anti-phospho-c-Fos (Ser32) (1:2000, Cell Signaling Technology) rabbit polyclonal antibodies were employed. Anti-vinculin mouse monoclonal antibody (1:2000, Merck) was used as loading control. Immunoreactivity was visualized with SuperSignal™ West Pico PLUS Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with iBright Analysis Software (Thermo Fisher Scientific).