Odyssey blocking buffer in pbs

Vero cell monolayers in six-well plates at 32°C were inoculated for two h with 250 PFU per well of virus, an overlay containing 0.8% methylcellulose was added to each well, and the cells were incubated for seven days and then fixed with 80% cold methanol. After overnight incubation in methanol, plates were incubated with a cocktail of three anti-RSV F mAbs [58 (link)] in Odyssey Blocking Buffer in PBS (Li-Cor), washed, and incubated with an R-phycoerythrin goat anti-mouse IgG(H+L) secondary antibody (Thermofisher). Plaques were visualized using the Celigo imager (Nexcelcom Bioscience). Images were analyzed using Celigo software to measure the plaque size (μm²) and the median fluorescence intensity (MFI) of F expression per plaque (i.e., the median value of all of the pixels per plaque). An average of 2140 (±1167) individual plaques were analyzed per virus in Fig 2B.

Materials and their respective vendors are as follows: DMEM, PBS, 200 mM l-Glutamine, 100× Penicillin/Streptomycin 0.25% Trypsin + 0.1%EDTA (Corning); Fetal Bovine Serum (Atlanta Biologicals); Fugene HD (Promega); Triton-X 100, DAPI (Roche); digitonin (ACROS); HEK 293T/17, COS-7, mouse anti-MYC 9E10.2 (conditioned media used at 1/10 dilution) (ATCC); 8-chamber glass slides, mouse anti-HA (1/2000 dilution) Immobilon-FL (Millipore), 16% paraformaldehyde (EMS); BSA, rabbit anti-FLAG (1/2000 dilution), DMSO, fatty acid free BSA, TCEP Tris(2-carboxyethyl) phosphine hydrochloride, TBTA Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, Copper Sulfate, NP-40 (Sigma); Alexa Fluor 647-AffiniPure Goat Anti-rabbit IgG (H + L) (1/200 dilution), Cy2 AffiniPure Donkey Anti-Mouse IgG (H + L) (1/200 dilution), anti-mouse Alkaline Phosphatase (H + L) (1/1500), anti-rabbit Alkaline Phosphatase (H + L) (1/1500) (Jackson ImmunoResearch); Mouse anti-GFP (1/15 000 dilution) (Clontech); SlowFade Gold antifade reagent (Thermo Scientific); Biotin Azide, Alexa Fluor_680 Goat Anti-mouse (1/4000 dilution), pcDNA3.1 (Invitrogen); IRDye800 conjugated streptavidin (1/5000 dilution), Odyssey blocking buffer in PBS (Licor); Protein A/G agarose beads, Halt protease and phosphatase inhibitor cocktail (Pierce); and Palmitic Acid Alkyne (Cayman Chemical).

Mammalian whole cell lysates were prepared by boiling 500,000 cells in 1x Sample Buffer (SB; 60 mM Tris-HCl [pH = 6.8], 5% glycerol, 2% SDS, 10% β-mercaptoethanol, 0.025% bromophenol blue, 1x Mammalian PI cocktail) for 5 min at 99°C. Sedimentation assay samples were prepared as described above. Western blot samples were boiled for 5 min at 99°C in 1x SB, separated by SDS-PAGE on a gradient gel (4%–20%, Bio-Rad CAT# 3450033), and transferred to a PVDF membrane. Membranes were blocked in Odyssey Blocking Buffer in PBS (Li-Cor CAT# 927–40000) for 1 hr at 25°C. Blots were then incubated in primary antibody overnight at 4°C and then in secondary for 30 min at 25°C. The antibodies used were: α-CLPB (Abcam CAT# ab235349), α-HAX1 (Abcam CAT# ab137613), α-COXIV (Abcam CAT# ab14744), α-GAPDH (Abcam CAT# ab8245), α-MICU2 (Abcam CAT# ab101465), IRDye 800CW Goat α-mouse secondary (Li-Cor CAT# 926–32210), and IRDye 680RD Goat α-rabbit secondary (Li-Cor CAT# 926–68071). Blots were imaged on a Li-Cor Odyssey Fc Imaging System.

Western blots were either performed from total cell lysates obtained by lysing cells directly with RIPA buffer complemented with PMSF, protease inhibitor cocktail and sodium orthovanadate (Santa Cruz, CAT# sc-24948). Proteins concentration was determined using BCA protein assay kit (Thermo). SDS-PAGE electrophoresis was carried out in 7.5% pre-cast polyacrylamide gels (Bio-Rad, CAT# 5671023). After transfer onto nitrocellulose, membranes were blocked for 30 min with Odyssey Blocking Buffer in PBS (Part Number: 927-40000, LI-COR Biosciences) and probed overnight with primary antibody: p-HSP27(Ser 82) (1:500, Cell Signaling, CAT# 9709), p-p38 (1:1000, Cell Signaling, CAT# 9211), p38 (1:1000, Cell Signaling, CAT# 8690), MyHC (1:500, Millipore, CAT# 05-716), GAPDH (1:500, Millipore, CAT# MAB374). After that, IRDye700-conjugated or IRDye800-conjugated secondary antibodies were used and visualized with the Odyssey infrared detector (LI-COR Biosciences, Westburg, Leusden, The Netherlands). For Protein quantification a, we used the background correction option in the software of the supplier (Image Studio™ Software for the Odyssey CLx-LI-COR Biosciences) and scanned the corresponding band of the protein of interest.

Whole cell lysates were extracted from cells in 2x sample buffer and separated on 10% SDS–PAGE gel and transferred to nitrocellulose membranes (GE Healthcare). The membranes were blocked with Odyssey Blocking Buffer in PBS (Licor), followed by incubation with primary antibodies against human FOXR1 (Biorbyt, rabbit 1:200), GFP (synaptic systems, mouse 1:1000), GAPDH (EMD Millipore, mouse 1:5000), HSPA1A and HSPA6 (Enzo life sciences, mouse 1:1000), DHRS2 (Abcam, rabbit 1:500), Histone H3 (Cell-Signaling, rabbit 1:1000) overnight at 4°C. Proteins recognized by the antibodies were detected with an Odyssey infrared imaging system (LI-COR) using IRDye680RD- or IRDye800CW-coupled secondary antibodies (LI-COR, 1: 20,000).