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Figure legends
Fig. 1. (A) Western blot analysis of E5 protein from three healthy and
four representative diseased bladder mucosa samples. Results of Western
blot analysis are representative of three independent experiments.
Fig. 2. Immunoprecipitation assay using anti-TRIM25 and anti-Riplet
antibodies in healthy and pathological bladder samples. Western blot
analysis revealed that TRIM25 only interacted with E5 protein. Panels A
and B show representative data from three independent experiments
Fig. 3. (A) Western blot analysis of total Riplet protein in healthy and
diseased bladder samples. (B) Densitometric analysis of total Riplet
protein relative to the β-actin protein level. Panels A and B show
representative data from three independent experiments.
Fig. 4. (A) Western blot analysis of TRIM25 in 10 normal and 15 diseased
bladder samples. (B) Densitometric analysis was performed by comparing
the protein expression level of TRIM25 with that of β-actin. TRIM25
protein level was significantly lower in pathological bladder mucosa
samples than in healthy samples. The calculations were based on two
independent determinations. The values are expressed as a percentage of
the average values for the healthy samples (**p ≤ 0.01).
Fig. 5: (A) Western blot analysis of RIG-1 and MDA5 in normal and
pathological bovine urinary bladder samples. (B) Densitometric analysis
was performed by comparing the protein expression levels of total RIG-1
and MDA5 with those of β-actin. RIG-1 and MDA5 protein levels were
significantly reduced in the infected mucosa samples compared with the
healthy samples (* p ≤ 0.05).
Fig. 6. Real-time RT-PCR analysis of RIG-I and MDA5 mRNA levels in 10
healthy and 15 pathological bladder samples. RIG-I and MDA mRNA
expressions were significantly reduced in diseased bladder samples
compared with normal bladder samples (*** p ≤ 0.001). Data are expressed
as the mean ± S.E.M. of three independent experiments performed in
triplicate.
Fig. 7. Real-time RT-PCR of TRIM25 mRNA levels in 10 healthy and 15
pathological bladder samples. Data are expressed as the mean ± S.E.M. of
independent experiments performed in triplicate.
Fig. 8. Immunoprecipitation using an anti-MAVS antibody in healthy and
diseased bladder samples. Western blot analysis revealed that MAVS
interacted with RIG-I, MDA5, TRIM25, phosphorylated TBK1 (pTBK1),
phosphorylated IRF3 (pIRF3) and Sec13. Immunoprecipitation panel shows
representative data from three independent experiments.
Fig. 9. (A) Western blot analysis of total Sec13 protein performed in
healthy and pathological bladder mucosa samples. (B) Densitometric
analysis was performed by comparing the protein expression level of
total Sec13 with that of β-actin. Sec13 protein level was significantly
reduced in the pathological bladder mucosa samples (** p ≤ 0.01). Panels
A and B show representative data from three independent experiments.
Fig. 10. (A) Western blot analysis of IRF3 and TBK1 in normal and
diseased bovine bladders. (B) Densitometric analysis was performed by
comparing the protein expression levels of total TBK1 and IRF3 with
those of β-actin. IRF3 and TBK1 protein levels were significantly
reduced in the neoplastic bladder mucosa samples compared with healthy
samples (*** p≤0.001 and * p ≤ 0.05, respectively). Panels A and B show
representative data from three independent experiments.
Fig. 11. (A) Western blot analysis of phosphorylated TBK1 (pTBK1) in the
total lysate in healthy and pathological samples. (B) Densitometric
analysis of pTBK1 protein was performed relative with β-actin protein
levels. The calculations were based on three independent determinations.
The values for the latter are expressed as percentages of the average
values for the healthy samples (* p≤0.05). Panels A and B show
representative data from three independent experiments.
Supplemental Fig S1. (A) Real-time RT-PCR analysis of BPV-2 and BPV-13
E5 mRNA expression in healthy and pathological bovine bladder samples.
Lane MW: DNA molecular weight marker (100-base pair (bp) ladder); lanes
2 – 4: three representative diseased bladder samples; lane 5: healthy
bladder sample; lane C: no template control (no cDNA added). (B) The
amplicon sequences showed 100% identity with BPV-2 E5 and BPV-13 E5
sequences deposited in GenBank (Accession numbers: M20219.1 and
JQ798171.1, respectively). Electrophoretic representative data were
obtained from three independent experiments.
Supplemental Fig S2. (A) TRIM25 cDNA amplification by PCR in normal and
pathological bovine urinary bladder samples compared with β-actin. Lane
1: molecular weight marker (DNA marker ladder); lanes 2-5: four
representative diseased bladder samples; lanes 6-9: healthy bladder
samples; in the last channel: negative control (RNA without reverse
transcriptase subjected to PCR analysis). (B) The lower part of the
figure shows the alignment of the sequences, which revealed 100%
identity with bovine TRIM25 transcript sequences deposited in GenBank
(Bos taurus tripartite motif containing 25 (TRIM25), mRNA:
NM_001100336.1).
Supplemental Fig S3. (A) RIG-I and MDA5 cDNA amplification by PCR in
normal and neoplastic bovine urinary bladder samples compared with
β-actin. Lane 1: molecular weight marker (DNA marker ladder); lanes 2-5:
four representative diseased bladder samples; lanes 6-9: healthy bladder
samples; in the last channel: negative control (RNA without reverse
transcriptase subjected to PCR analysis).
(B) The lower part of the figure shows the alignment of the sequences,
which revealed 100% identity with bovine RIG-I and MDA5 transcript
sequences deposited in GenBank (Bos taurus DExD/H-box helicase 58
(DDX58), transcript variant X1, mRNA: XM_002689480.6; Bos taurus
interferon induced with helicase C domain 1 (IFH1), mRNA:
XM_010802053.2).