Thyroid peroxidase gene expression is induced by lipopolysaccharide involving Nuclear Factor (NF)-κB p65 subunit phosphorylation
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1 SUPPLEMENTAL DATA Thyroid peroxidase gene expression is induced by lipopolysaccharide involving Nuclear Factor (NF)-κB p65 subunit phosphorylation Magalí Nazar, Juan Pablo Nicola, María Laura Vélez, Claudia Gabriela Pellizas, Ana María Masini- Repiso. SUPPLEMENTAL TABLE 1: Primers and probes information A. Primer sets used for RT-qPCR and ChIP assays Designation Primer Sequence (5 to 3 ) TPO (F) GCATGTATCATTGGGAAGCA (R) CGGTGTTGTCACAGATGACC Amplicon Size (bp) Intron Spanning Sequences (bp) qpcr Efficiencies % β-actin (F) GGCACCACACTTTCTACAATG (R) TGGCTGGGGTGTTGAAGGT % ChIP P1 (F) GGAGCGAGAACTGGTGAAGG (R) CTAGGGTCACCCACATAGGTTC 92 (-369/-277 # ) 101% ChIP P2 (F) CTGCTTTCTATGAGTGGCACC (R) CTCTCTGGAGACTTGGTTACC 163 (-163/-53 # ) 99% B. Primer sets used for construction of ptpo κb Primer Sequence (5 to 3 ) (F) AGGGTACCAGACTGCTTTCTA (R) TCTGCTAGCCACACAGACCAGTAATG C. Primers used for Site-directed Mutagenesis for construction of ptpo κbm Primer Sequence (5 to 3 ) (F) TGGGATGAACTAGAAATATAGGATAAGATAAACACACAGGAACC TATGTGGGTG (R) CACCCACATAGGTTCCTGTGTGTTTATCTTATCCTATATTTCTAGT TCATCCCA 1
2 D. Oligonucleotides used for EMSA Designation Primer Sequence (5 to 3 ) Location TPO κb TPO κbm TPO Z TPO Zm TPO B TPO Bm (F) GGATAAGAGAAACTCCCAGGAACC (R) gctatggttcctgggagtttctcttatcc (F) GGATAAGGAGAACCTTCAGGAACC (R) gctatggttcctgaaggttctccttatcc (F) ACAAATACTAAACAAACAGAATGG (R) gctatccattctgtttgtttagtatttgt (F) ACAATAATCAAACAACAGAATGG (R) gctatccattctgttgtttgattattgt (F) CACACAAGCACTTGGCAGAAACAA (R) gctatttgtttctgccaagtgcttgtgtg (F) CACACAAGCACGGTCCAGAAACAA (R) gctatttgtttctggaccgtgcttgtgtg -318/-294 # -155/-131 # -175/-151 # # Positions given are relative to TPO transcription start site. Shaded nucleotides represent introduced desired mutations. Lower case sequences represent a non-native short 3 overhang addition for α- 32 P-dATP fill-in labeling reaction mediated by Klenow (EMSA assays). Underlined sequences indicated restriction enzimes sites used for subcloning into pgl3-basic. 2
3 Supplemental Figure 1. LPS increases TSH-induced binding of TTF-1 and TTF-2 to B and Z sites in the TPO promoter. Nuclear proteins were isolated from FRTL-5 cells treated with TSH (0.5 miu/ml) or TSH + LPS (100 ng/ml) for 1 h. EMSA was performed with 32 P-labeled oligonucleotide corresponding to the TTF-1 binding site B (probe TPO-B) or TTF-2 consensus site Z (probe TPO-Z) described in the TPO promoter. A, Representative EMSA assay demonstrating that LPS increased 3
4 TSH-induced TTF-1 recruitment to region B (lanes 2 and 5). Specificity was tested by performing competition experiments in the presence of a 100-fold excess of cold TPO-B (lanes 3 and 6). Incubations done in the presence of an anti-ttf-1 antibody decreased shifted complex (lanes 4 and 7). B, Representative EMSA assay showing that LPS increased TSH-induced TTF-2 binding to region Z (lanes 2 and 5). Band shift specificity was demonstrated by conducting competition assays with 100-fold excess of cold oligonucleotide Z (lanes 3 and 6). The presence of TTF-2 in the shifted complex was accomplished using an anti-ttf-2 antibody (lanes 4 and 7). NS, nonspecific. 4
5 Supplemental Figure 2. LPS increases TTF-1 and TTF-2 nuclear levels. Starved FRTL-5 cells were treated with TSH (0.5 miu/ml) in presence or absence of LPS (100 ng/ml) for 1h and then harvested for nuclear-cytoplasmic protein preparation. Representative Western blot of cytoplasmic and nuclear extracts (40μg) showing TTF-1 and TTF-2 levels. Nuclear recruitment of p65 was evaluated as positive control of LPS action. Histone H1 was used to correct loading differences in nuclear extracts. α-tubulin was assess to exclude cytoplasmic contaminants in nuclear extracts. 5
6 Supplemental Figure 3. p38 activation does not affect p65 nuclear translocation. A, Representative Western blot of whole proteins obtained from FRTL-5 cells treated after starvation as indicated in the figure for 30 min. Histone H1 staining was used to correct loading differences. Densitometric analysis was carried out to determine the relative nuclear level increase of p65 normalized to Histone H1. B, Relative activity of an artificial NF-κB-responsive vector containing five κb sites in tandem linked to Luc (5x κb-luc) after the indicated stimulus for 24 h in presence or absence of p38 inhibitor (SB μm), which was added to the culture medium 1 h before treatment. Results are expressed as Luc activity normalized to β-galactosidase and relative to basal activity for each construct. #, P < 0.05 vs. basal; **, P < 0.01 vs. TSH (ANOVA; Student-Newman- Keuls). 6
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Supporting Online Material for
www.sciencemag.org/cgi/content/full/1154040/dc1 Supporting Online Material for Selective Blockade of MicroRNA Processing by Lin-28 Srinivas R. Viswanathan, George Q. Daley,* Richard I. Gregory* *To whom
Supplemental Data. Sun et al. Plant Cell. (2012) /tpc FLS2 -cmyc -GFP FLS2 -HA. FLS2-FLAG FLS2 -HA BAK1-HA flg22 (10mM)
Supplemental Data. Sun et al. Plant ell. ()..5/tpc..9599 A FLS-FLA FLS -HA BAK-HA flg (mm) - B FLS -cmyc -FP FLS -HA IP antibody cmyc FLA FP cmyc IP ; WB: α-ha IP; WB: α-ha IP: α-fla; WB: α-ha IP ; WB:
PrimePCR Assay Validation Report
Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID protein kinase N1 PKN1 Human The protein encoded by this gene belongs to the protein
Supplemental Data. Hu et al. Plant Cell (2017) /tpc
1 2 3 4 Supplemental Figure 1. DNA gel blot analysis of homozygous transgenic plants. (Supports Figure 1.) 5 6 7 8 Rice genomic DNA was digested with the restriction enzymes EcoRⅠ and BamHⅠ. Lanes in the
Khaled_Fig. S MITF TYROSINASE R²= MITF PDE4D R²= Variance from mean mrna expression
Khaled_Fig. S Variance from mean mrna expression.8 2 MITF.6 TYROSINASE.4 R²=.73.2.8.6.4.2.8.6.4.2.8.6.4.2 MALME3M SKMEL28 UACC257 MITF PDE4D R²=.63 4.5 5 MITF 3.5 4 PDE4B 2.5 3 R²=.2.5 2.5.8.6.4.2.8.6.4.2
Supplementary Fig.1. Over-expression of RNase L in stable polyclonal cell line
Supplemental Data mrna Protein A kda 75 40 NEO/vector NEO/RNase L RNASE L β-actin RNASE L β-actin B % of control (neo vector), normalized 240 ** 200 160 120 80 40 0 Neo Neo/RNase L RNase L Protein Supplementary
SUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb2271 Supplementary Figure a! WM266.4 mock WM266.4 #7 sirna WM266.4 #10 sirna SKMEL28 mock SKMEL28 #7 sirna SKMEL28 #10 sirna WM1361 mock WM1361 #7 sirna WM1361 #10 sirna 9 WM266. WM136
Post-translational modification
Protein expression Western blotting, is a widely used and accepted technique to detect levels of protein expression in a cell or tissue extract. This technique measures protein levels in a biological sample
Supplemental Figure 1.
Supplemental Data. Charron et al. Dynamic landscapes of four histone modifications during de-etiolation in Arabidopsis. Plant Cell (2009). 10.1105/tpc.109.066845 Supplemental Figure 1. Immunodetection
Construction of plant complementation vector and generation of transgenic plants
MATERIAL S AND METHODS Plant materials and growth conditions Arabidopsis ecotype Columbia (Col0) was used for this study. SALK_072009, SALK_076309, and SALK_027645 were obtained from the Arabidopsis Biological
RayBio Human FRA-2 Transcription Factor Activity Assay Kit
RayBio Human FRA-2 Transcription Factor Activity Assay Kit Catalog #: TFEH-FRA2 User Manual May 2, 2017 3607 Parkway Lane, Suite 200 Norcross, GA 30092 Tel: 1-888-494-8555 (Toll Free) or 770-729-2992,
Supplemental material
Supplemental material THE JOURNAL OF CELL BIOLOGY Taylor et al., http://www.jcb.org/cgi/content/full/jcb.201403021/dc1 Figure S1. Representative images of Cav 1a -YFP mutants with and without LMB treatment.
Supplementary Figure 1. Intracellular distribution of the EPE peptide. HeLa cells were serum-starved (16 h, 0.1%), and treated with EPE peptide,
Supplementary Figure 1. Intracellular distribution of the EPE peptide. HeLa cells were serum-starved (16 h, 0.1%), and treated with EPE peptide, conjugated with either TAT or Myristic acid and biotin for
SUPPLEMENTARY INFORMATION
doi:10.1038/nature11070 Supplementary Figure 1 Purification of FLAG-tagged proteins. a, Purification of FLAG-RNF12 by FLAG-affinity from nuclear extracts of wild-type (WT) and two FLAG- RNF12 transgenic
Data Sheet GITR / NF-ĸB-Luciferase Reporter (Luc) - Jurkat Cell Line Catalog #60546
Data Sheet GITR / NF-ĸB-Luciferase Reporter (Luc) - Jurkat Cell Line Catalog #60546 Description This cell line expresses a surface human GITR (glucocorticoid-induced TNFR family related gene; TNFRSF18;