Rabbit anti alpha tubulin

Thirty micrograms of protein was separated on an 8% SDSPAGE gel and transferred to polyvinylidenedifluoride membranes (Millipore). Membranes were blocked and then incubated for 2 hours with either rabbit anti-alpha Tubulin (1:1000, Abcam), mouse anti-CD63 (1:1000, Abcam) or mouse anti-TSG101 (1:200, Abcam). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG was used as a secondary antibody (diluted 1:5,000 in TBST). Bands were scanned using a densitometer (GS-700; Bio-Rad Laboratories).

After the exposure, intestinal organoids were lysed in 100 μL RIPA buffer and the extracted proteins were denatured using SDS loading buffer (125 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.04% bromophenol blue, and 100 mM β-mercaptoethanol) at 95 °C for 5 min. For each sample, 10 μg protein was loaded and separated on 10% or 12% mini-protein TGX precast protein gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chicago, USA). The membranes were incubated overnight at 4 °C with appropriate primary antibodies, subsequently with specific secondary antibodies and visualized using the enhanced chemiluminescence reagent (Thermo Scientific). The following antibodies were used: Rabbit anti-alpha-TUBULIN, mouse anti-E-cadherin, and rabbit anti-NF-κB p65 were from Abcam; rabbit antibodies against p38, phospho-p38, JNK, phospho-JNK, ERK, phospho-ERK, MLC, phosphor-MLC, phospho-NF-κB p65, STAT1, and phospho-STAT1 (Tyr701) were from Cell Signalling Technology; mouse anti-CLDN-2 and rabbit anti-ILDR-1 were from Invitrogen; anti-mouse/rabbit IgG horseradish peroxidase-linked secondary antibodies were from Cell Signalling Technology. Original western blot images were included in Supplementary Figures 7, 8, and 9. Densitometric quantification analyses of the western blots were performed using the Image J software.

To analyse viral gene expression from transfected plasmids, a previously published strategy was adopted (Wise et al., 2011 (link)). Briefly, nucleotides 1–380 of UDL-1/08 PB1 were cloned into AgeI/KpnI sites of pEGFPN1 (Clontech). Using site-directed mutagenesis the position of the EGFP ORF was adjusted into frame with either the PB1 or PB1-F2 reading frames, while concurrently removing the GFP ATG codon. Additional segment 2 mutations as detailed in the results section were made using site directed mutagenesis with the WT segment plasmids as templates. 2 µg of each plasmid was transfected into HEK 293T cells using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 12 h after transfection, 10 µM MG132 (Sigma-Aldrich) was added to the medium for a further 12 h. Cells were harvested and the presence of alpha-tubulin and PB1-, PB1-N40- and PB1-F2-GFP was detected via Western blot using rabbit anti-alpha tubulin (Abcam) and mouse anti-GFP (Gene Tex) antibodies with IRDye 800CW/IRDye 680RD secondary antibodies (LI-COR) and visualized using the Odyssey CLx (Li-Cor). Densitometric analysis was performed using Image Studio software (Li-Cor) from three independent experiments and the intensities for PB1-, PB1-N40- and PB1-F2-GFP were normalized to alpha-tubulin. Fold change in protein expression relative to the WT was calculated.