Skimmed milk powder

Eighty micrograms of primary epithelial cell lysates or 1 µg purified receptor candidates separated in SDS-PAGE gels were Western-blotted onto nitrocellulose (Amersham, GE Healthcare) with Schaeffer–Nielsen buffer [48 mM Tris, 39 mM glycine, 20 % (v/v) methanol, 0.04 % (v/v) SDS, pH 9.2], at 15V for 30–60 min. Blots were blocked in Carbofree (Vector Labs) or 0.1 % (v/v) Tween₂₀ (Sigma) in PBS (PBST) with 5 % (w/v) skimmed milk powder (Sigma, Marvel) at RT for 2 h or 4 °C for 16 h. Blots were washed three times between all subsequent steps in PBST alone for 15 min at RT, with rocking. For both Western and far-Western blots, antibodies were diluted in 1 % (w/v) skimmed milk powder in PBST and incubated with blots for 1 h, with rocking at RT.
Prior to antibody labelling, far-Western blots were first probed with 1 µg ml⁻¹ H7 flagella in PBS for 3 h, with rocking at RT. H7 flagella were then treated with α-H7 rabbit IgG diluted 1 : 1000, and this was labelled with horseradish peroxidase (HRP)-conjugated α-rabbit IgG diluted 1 : 1000. Western blots of cell lysates were probed with α-cofilin-1 mouse IgG diluted 1 : 500 or α-galectin-4 goat IgG diluted 1 : 1000, and then a 1 : 1000 dilution of HRP-conjugated α-mouse or goat IgG, respectively. Blots were then developed using Pico-West SuperSignal ECL reagents (Thermo Fisher) in a G:box (Syngene), and captured using GeneSnap (Syngene).

Cell wall extracts and bacterial supernatants were prepared as described previously [50 (link)] with minor modifications. Briefly, stationary phase cultures were pelleted and supernatants were 0.2 μm filtered. Cell pellets were incubated in cell wall extraction buffer (30% raffinose, 1 μg/ml lysozyme, 10 mM Tris-HCl (pH 8)) at 37°C, 3 hours and cell wall fraction was isolated by centrifugation. Raffinose was removed following dialysis into PBS using Slide-A-lyzer cassettes (ThermoFisher Scientific), and concentrated to 100 μg/ml. 1 μg cell wall extract was loaded for each sample to allow direct comparison between strains. Samples were separated on 4–12% Bis-Tris plus gels (ThermoFisher Scientific) and transferred onto Hybond-LFP membrane (Amersham). Membranes were blocked (5% skimmed milk powder (Sigma), 0.05% Tween (Sigma) and probed and detected with relevant primary and secondary antibodies. Membranes were developed using ECL Advance western blotting detection system (GE Healthcare).

Sodium citrate tribasic dehydrate (C₆H₅Na₃O₇·2H₂O), sodium chloride (NaCl), sodium hydroxide (NaOH), potassium chloride (KCl), bovine serum albumin (BSA), potassium dihydrogen orthophosphate (KH₂PO₄), uranyl acetate, and tris base (C₄H₁₁NO₃) were obtained from Sisco Research Laboratories (SRL, India). Sodium dihydrogen phosphate-1-hydrate (Na₂HPO₄·H₂O) and sodium tetraborate (Na₂B₄O₇) were purchased from Merck (Mumbai, India). Gold(iii) chloride (Au₂Cl₆), boric acid (H₃BO₃), and skimmed milk powder were procured from Sigma Aldrich (India). Hydrochloric acid (HCl) and ethanol were procured from Fisher Scientific (India), while anti-rabbit immunoglobulin G (IgG) antibodies were purchased from G-Biosciences (Noida, India). Dengue-2 Virus, West Nile Virus, and Yellow Fever Virus NS1 proteins were acquired from The Native Antigen Company (Oxfordshire, UK). The Japanese Encephalitis Virus NS1 Ag and Ab were developed in the lab as explained in earlier work.^{14 (link)} JEV positive and negative pig serum real samples were a kind gift from the ICAR-Indian Veterinary Research Institute (IVRI) (Uttar Pradesh, India). All high analytical grade reagents were used and all solutions were prepared in double distilled water unless mentioned otherwise.

For the filter retardation assay, 12.5 μg of protein homogenate was diluted in Dulbecco's phosphate‐buffered saline (DPBS; Gibco) containing 2% sodium dodecyl sulphate (SDS) and 50 mM 1,4‐dithiothreitol (DTT) and heat‐denatured for 5 min at 95°C. Samples were filtered through Amersham Protran Premium 0.45‐μm nitrocellulose membranes (GE Healthcare) by using a Minifold II Slot‐Blot System (Schleicher & Schuell). Before loading the samples, the membrane was equilibrated with DPBS containing 0.1% SDS and rinsed afterwards twice with DPBS. The membranes were blocked in tris‐buffered saline (TBS; 1 M Tris, 5 M NaCl) containing 5% skimmed milk powder (Sigma‐Aldrich) for 1 h, followed by primary antibody (mouse anti‐ataxin‐3, 1:2,500, clone 1H9, MAB5360, Merck Millipore) incubation overnight at 4°C, and secondary antibody (IRDye 800CW goat anti‐mouse IgG (H+L) 1:1,000, 926‐32210, LI‐COR) incubation for 1.5 h at room temperature. Detection and quantification of the fluorescent signal was performed using the ODYSSEY Server software version 4.1 (LI‐COR Bioscience).

Cells and tissue were first lysed with RIPA buffer (Beyotime Biotechnology, Shanghai, China) at room temperature for 1 h. The bicinchoninic acid method was used to measure the level of proteins. Proteins in the lysate were separated by electrophoresis and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skimmed milk powder (Sigma-Aldrich) and then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: anti-MEX3C (Cell signaling, #50,844, 1:1,000), anti-RUNX3 (Cell signaling, #9647, 1:1,500), anti-Suv39H1 (Novus, NBP1-21,367, 1:1,000), anti-E-cadherin (Abcam, ab231303, 1:1,500), anti-N-cadherin (Abcam, ab76011, 1:1,000), anti-Bcl-2 (Abcam, ab182858, 1:1,000), anti-Bax (Abcam, ab32503, 1:1,000), anti-Cleaved-caspase-3 (Cell signaling, #9661, 1:1,000), and anti-GAPDH (Abcam, ab8254, 1:2,000). The membranes were then washed in TBST and incubated with horseradish peroxidase-labeled secondary antibody. After washing in TBST again, protein bands were visualized using an ECL detection agent (GE Healthcare, Chicago, IL, USA) and ImageJ software. GAPDH were used as internal loading controls.