Tween 40

3′ RACE and sequence analysis of antigenome and genome RNA (Figure 8) was performed using RNA isolated from infected cell extracts enriched for RSV ribonucleoprotein (RNP) complexes [75] (link). Briefly, 8×10⁶ HEp2 cells were infected at an MOI of 5. At 17 h post infection, the supernatant was replaced with media containing 2 µg/ml actinomycin D and cells were incubated at 37°C for a further 1 h. Following an ice cold PBS wash, cells were treated for 1 min with PBS supplemented with 250 µg/ml lyso-lecithin, on ice. Cells were scraped into 400 µl of ice cold Buffer A (50 mM tris-acetate pH 8, 100 mM K-acetate, 1 mM DTT, 2 µg/ml actinomycin D), disrupted by repeated passage through an 18G needle and incubated on ice for 10 min. Following centrifugation at 2400× g for 10 min at 4°C, the resulting pellet was disrupted in 200 µl of ice cold Buffer B (10 mM tris-acetate pH 8, 10 mM K-acetate, 1.5 mM MgCl₂, 1% triton X-100) by repeated passage through an 18G needle and then incubated on ice for 10 min. The sample was centrifuged and the resulting pellet was disrupted in 200 µl of Buffer B supplemented with 0.5% deoxycholate, 1% tween 40 as described above. Following a 10 min incubation on ice and a repeat centrifugation, the supernatant enriched for viral RNPs was collected and RNA was extracted using Trizol (Invitrogen). The purified RNA was tailed with either A or C residues using E. coli poly A polymerase (NEB), followed by heat inactivation of the enzyme, according manufacturer's instructions. First strand cDNA synthesis was performed using primers 5′ GAGGACTCGAGCTCAAGCATGCATTTTTTTTTTTTTTT, or 5′ GAGGACTCGAGCTCAAGCATGCATGGGGGGGGGGGGGGG, which hybridized to the poly A or poly C tail, respectively, and Sensiscript reverse transcriptase (Qiagen), according to manufacturer's instructions. To determine the sequence of the antigenome 3′ terminus, purified cDNA was PCR amplified using primer SLNQi 5′-GAGGACTCGAGCTCAAGC and a TrC specific primer Tr1: 5′-GCAGCACTTTTAGTGAACTAATCC. The resulting product was subjected to a second round of hemi-nested PCR using primer SLNQi and primer Tr2: 5′-GCAGTCGACCATTTTAATCTTGGAG. PCR products were gel purified and either sequenced directly or cloned into a pGEM vector for sequencing of individual cDNA clones. Analysis of the genome 3′ terminus was performed as described above, using the same cDNA preparation and primer SLNQi, but with NS1 specific primers: 5′-GCACAAACACAATGCCATTC and 5′-GCAGTCGACGTATGTATCACTGCCTTAGCC.

DPPH radical scavenging assay: The hydrogen atom or electron donation abilities of the extracts, along with the four fractions separated from the ethanol extracts (petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and water fraction) and the pure compounds were measured from the bleaching of a purple-colored ethanol solution of DPPH. This spectrophotometric assay uses the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a reagent [41 (link)]. An aliquot of the sample (100 µL) was mixed with ethanol (1.4 mL) and then added to 0.004% DPPH (1 mL, Sigma-Aldrich) in ethanol. The mixture was shaken vigorously and then immediately placed in a UV-Vis spectrophotometer (UNICO, Shanghai, China) to monitor the decrease in absorbance at 517 nm. Monitoring was continued for 70 min until the reaction reached a plateau. Ascorbic acid (Sigma-Aldrich), a stable antioxidant, was used as a synthetic reference. The radical-scavenging activities of samples, expressed as percentage inhibition of DPPH, were calculated according to the formula: Inhibition percentage (Ip) = [(AB-AA)/AB]×100 [42 ] where AB and AA are the absorbance values of the blank sample and of the tested samples checked after 70 min, respectively.
β-carotene-linoleic acid test: Antioxidant activity of the samples was determined using the β-carotene-linoleic acid test [43 (link)]. Approximately 10 mg of β-carotene (type I synthetic, Sigma–Aldrich) was dissolved in chloroform (10 mL). The carotene–chloroform solution, (0.2 mL) was pipetted into a boiling flask containing linoleic acid (20 mg, Sigma–Aldrich) and 200 mg Tween^® 40 (Sigma–Aldrich). Chloroform was removed using a rotary evaporator (RE-52AA) at 40 °C for 5 min, and distilled water (50 mL) was added to the residue slowly with vigorous agitation, to form an emulsion. A portion of the emulsion (5 mL) was added to a tube containing the sample solution (0.2 mL) and the absorbance was immediately measured at 470 nm against a blank, consisting of an emulsion without β-carotene. The tubes were placed in a water bath at 50 °C and the oxidation of the emulsion was monitored spectrophoto-metrically by measuring absorbance at 470 nm over a 60 min period. Control samples contained 200 µL of water instead. Butylated hydroxytoluene (BHT, Sigma–Aldrich), a stable antioxidant, was used as a synthetic reference. The antioxidant activity was expressed as inhibition percentage with reference to the control after a 60 min incubation using the following equation: AA = 100(DR_C -DR_S)/DR_C, where AA = antioxidant activity; DR_C = degradation rate of the control = [ln(a/b)/60]; DR_S = degradation rate in presence of the sample = [ln(a/b)/60]; a = absorbance at time 0; b = absorbance at 60 min.

Approximately 10 × 10⁶ U2OS cells were collected through trypsinization and washed twice in cold PBS 1x. The cell pellet was resuspended in 1 mL of lysis buffer [10 mM Tris–HCl (pH 8–8.4) 0.14 M NaCl (Merck), 1.5 mM MgCl₂ (Sigma-Aldrich) and 0.5% (v/v) NP-40 (Sigma-Aldrich)], before collection of 100 μL of the resulting lysates to be labeled as the whole-cell RNA fraction. The nuclei were spun down at 1 000 g for 4 min. The supernatant, representing the cytoplasmic fraction, was collected and centrifuged at 11 000 g for 1 min to remove the remaining nuclei. Nuclei pellets were resuspended in 1 mL of lysis buffer and 1/10 volume (100 μL) of a detergent solution [3.3% (w/v) sodium deoxycholate (Sigma-Aldrich) and 6.6% (v/v) Tween 40 (Merck)] under slow vortexing. The stripped nuclei were then centrifuged at 1 000 g, for 4 min at 4 °C. The nuclei pellet was then washed once more in lysis buffer and spun down at 1 000 g, for 4 min at 4 °C. The pellet enriched in nucleic content was resuspended in 1 mL of TRIzol using a 21-gauge syringe. TRIzol was also added to the whole-cell RNA and cytoplasmatic fraction and RNA was extracted according to the manufacturer’s instructions.

(i) Membrane-anchored LRP6. P114-L2/L-HA cells were seeded in 15 cm tissue culture dishes. The next day, cells were washed with cold PBS and lysed in RIPA buffer containing protease inhibitor (cOmplete mix, Roche). The cell lysates were transferred into tubes, passed through a 26 G size needle (0.45 × 25 mm BL/LB, B Braun), and centrifuged at 4°C at 3,200 × g for 20 min. The supernatants were transferred into new tubes. In separate tubes, anti-TST Ab (THE NWSHPQFEK-tagged mouse MAb, A01732, GenScript, 1:150) was mixed with 20 μL of magnetic beads (Dynabeads Protein G, Invitrogen) in 300 μL of PBS containing 0.05% Tween 40 (PBS-T, Merck) and was incubated at 4°C for 10 min. Next, 15 μg of soluble HOP-TST, Hwt, HOP^A393T-TST, and Hwt^R529A-TST proteins were added to the Dynabeads/Ab mixture and incubated for 30 min on a rotating wheel at 4°C. The H protein/Ab/Dynabeads complexes were then mixed with the harvested supernatants and incubated on a rotating wheel overnight at 4°C. After five washes with PBS containing 0.05% Tween 40 (PBS-T, Merck), the bead samples were suspended in 40 μL of Laemmli buffer (2×) supplemented with 200 mM DTT and boiled at 95°C for 10 min. The samples were subjected to Western blot analyses using 4 to 8% Tris-Acetate gel (Invitrogen) either by using an anti-HA high-affinity rat MAb (3F10, Roche; 1:2000, overnight) followed by incubation with a HRP-conjugated goat anti-rat antibody (Abcam; ab97057; 1:3000, 2 h) or by using an anti-TST Ab (THE NWSHPQFEK-tagged mouse MAb, A01732, GenScript; 1:2000, overnight) followed by a HRP-conjugated polyclonal goat anti-mouse Ab (P0447, Dako; 1:3000, 2 h).
(ii) Soluble LRP6. 2 μg of purified solLRP6-Fc or solSLAM.V-Fc were mixed with 2 μL of magnetic beads (Dynabeads Protein G, Invitrogen) in 300 μL of PBS containing 0.05% Tween 40 (PBS-T, Merck) using Protein LoBind tubes (L200956G, Eppendorf) and incubated on a rotating wheel overnight at 4°C. Then, 0.5 μg of soluble HOP-TST, Hwt, HOP^A393T-TST, and Hwt^R529A-TST (or no proteins for the control experiments) were added to each bead sample and incubated on a rotating wheel for 2 h at 4°C. After five washes with PBS containing 0.05% Tween 40 (PBS-T, Merck), the bead samples were resuspended in 10 μL of Laemmli SDS reducing (4×) sample buffer (J60015, Alfa Aesar) supplemented with 200 mM DTT and were boiled at 95°C for 10 min. Finally, the samples were subjected to Western blot analyses (using 10% Bis-Tris gels [GenScript]), as described above for the membrane-anchored LRP6 coimmunoprecipitation. A HRP-conjugated, goat anti-human IgG antibody (AP113P, Merckmillipore, 1:2000, overnight) was used to reveal the Fc proteins.