Anti tubulin antibody

Transfected cells were treated with 10 nM E2. One hour prior to harvest, cells received a second E2 treatment, and were lysed in RIPA buffer containing protease and phosphatase inhibitors. Cell lysates were then centrifuged at 10 000g for 20 min to remove debris. Protein concentrations were measured using a BCA protein assay kit (Pierce). Proteins separated by SDS–PAGE were transferred onto nitrocellulose membranes. After blocking with blocking buffer (Tris-buffered saline, 0.05% Tween 20, 5% dry skim milk, pH 7.4) for 1 h at room temperature, membranes were incubated for 24 h with phospho-ERK antibody at 1 : 1000 dilution (Cell Signaling Technology, Beverly, MA). After incubation with secondary antibodies conjugated with HRP, proteins were visualized using an ECL detection kit (Pierce) according to the manufacturer's instructions. The membranes were then stripped with a stripping buffer (Pierce) and re-probed with total ERK antibody (Cell Signaling no. 9102). To control for total protein loading, membranes were also probed with an anti-tubulin antibody (Abcam). We used the following catalogue numbers and vendors:

The cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet-P40, 0.5% SDS, 10% glycerol, 1 mM dithiothreitol [DTT], 1 mM NaF, and ethylenediaminetetraacetic acid-free mini protease inhibitor cocktail [catalog no. 11873580001, Roche], pH 8.0) and scraped off the dish. The lysates were briefly incubated on ice, passed three times through a 25-gauge needle, incubated on ice for 15 min, and centrifuged at 20,000 × g for 30 min at 4 °C. For Western blot, the supernatant samples were mixed with sample buffer and boiled for 5 min. The proteins were then resolved by SDS-PAGE and transferred to nitrocellulose membranes (catalog no. 66485, Pall Corporation) using Trans Blot Turbo (catalog no. 1704270, Bio-Rad). Membranes were blocked with 5% milk in TBST (20 mM Tris-HCl, 150 mM NaCl [pH 7.6], and 0.1% Tween20) and probed with anti-AChR-α1 antibody (catalog no. 10613-1-AP, Proteintech), anti-rapsyn antibody (catalog no. ab156002, Abcam) or anti-tubulin antibody (catalog no. ab18251, Abcam) in 5% milk in TBST. After washing with TBST buffer, the membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase. For protein detection, we used Clarity chemiluminescent substrate (catalog no. 1705060, Bio-Rad).

ARVM and HL-1 cells were treated as indicated and lysed in buffer containing 50 mM HEPES (pH 7.4) (PromoCell, Heidelberg, Germany), 1 % Triton X-100 (Sigma-Aldrich, Munich, Germany), PhosSTOP and CompleteTM protease inhibitor cocktail (Roche, Basel, Switzerland). After incubation for 2 h at 4 °C, the suspension was centrifuged at 10,000g for 15 min. 5 μg protein sample of the total cell lysate was separated by SDS/PAGE (10 % gel) and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in tris-buffered saline (TBS) containing 0.1 % tween 20 and 5 % (w/v) non-fat dried skimmed milk powder and incubated overnight with anti-phospho Akt(Ser⁴⁷³) antibody, anti-phospho Akt(Thr³⁰⁸), anti-GAPDH antibody (all Cell Signalling Technology, Danvers, MA, USA) or anti-tubulin antibody (Abcam, Cambridge, UK). After washing, membranes were incubated with appropriate horseradish peroxidase-coupled secondary antibody and processed for enhanced chemiluminescence (ECL) detection using Immobilion horse radish peroxidase (HRP) substrate (Millipore, Darmstadt, Germany). Signals were visualised and evaluated on a VersaDoc 4000 MP Bio-Rad Laboratories work station and analysed by Quantity One analysis software (version 4.6.7) (both Bio-Rad Laboratories, Hercules, CA, USA).

To evaluate the macrophage-platelet interaction during DBV infection, 8 × 10⁵ PMA-activated THP-1 cells were seeded onto a confocal plate (Biosharp, Anhui, China) and infected by DBV (MOI = 1). Macrophages treated with LPS and nigericin or untreated were served as positive and negative controls, respectively. After 72 h incubation, macrophages were washed three times with precooled PBS, fixed with 1% paraformaldehyde for 5 min, and penetrated with 0.1% Triton-X for 10 min. After blocking with immunostaining blocking solution (Beyotime, Shanghai, China) for 10 min, samples were incubated overnight at 4°C with anti-tubulin antibody (1:200) (Abcam, Britain), anti-caspase-1 antibody (1:200) (Santa Cruz Biotechnology, USA) and anti-DBV-Gc antibody (1: 200) (Sangon Biotech, Shanghai, China). Samples were washed with PBS and incubated with donkey anti-rabbit IgG H&L (Alexa Fluor 568) pre-adsorbed (1:200) (Abcam, Britain), goat anti-mouse IgG H&L (Alexa Fluor 488) pre-adsorbed (1:200) (Abcam, Britain) and donkey anti-rat IgG H&L (Alexa Fluor 647) pre-adsorbed (1:200) (Abcam, Britain) for 1 h. The nuclei were then stained using the mounting medium with DAPI (Abcam, Britain). The images were obtained using a laser confocal microscope (ZEISS, LSM880, Germany).

Rat kidney samples were lysed with RIPA lysis buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors. The protein concentration was determined by the BCA assay (Thermo Fisher, Waltham, MA, USA). The proteins in these extracts were separated by electrophoresis and transferred onto a nitrocellulose membrane (Bio-Rad, Heracles, CA, USA), which was next blocked with 1% BSA for 1 h and then incubated overnight at 4 °C with a relevant primary antibody: anti-E-cadherin antibody, anti-vimentin antibody, anti-α-smooth muscle actin (SMA) antibody, anti-peroxisome proliferator-activated receptor alpha (PPARA) antibody, anti-PPARG antibody, anti-tumor necrosis factor-alpha (TNF-α) antibody, anti-transforming growth factor-beta (TGF-β1) antibody, anti-tubulin antibody, and anti-GAPDH antibody (Abcam, Eugene, OR, USA; cat. # ab231303, ab92547, ab7817, ab227074, ab178860, ab205587, ab179695, ab7291, and ab845, respectively). After an immunoreaction with a corresponding secondary antibody (Abcam, Boston, MA, USA, cat. # ab6721), bands of proteins were detected by means of a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). GAPDH and tubulin were employed as the internal loading controls.