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Published byBrandon Brown Modified over 7 years ago
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Figure S1. Production of recombinant NS1 protein
Figure S1. Production of recombinant NS1 protein. The nucleotide sequence of the NS1 protein from DENV-2, strain 16681, was cloned into the pCR 2.1 TOPO vector. Next, the sequence was subcloned into the BamH1 and Xho I sites of the expression vector pPROEX HTb. A) pPROEX HTb multiple cloning site. The 5’ end of the NS1 was inserted into the BamHI site and the 3’ end in the XhoI site. B) The presence of the NS1 sequence in the recombinant plasmid pPROEX-NS1 was confirmed by the release of a 1055 bp fragment after digestion with BamHI and XhoI enzymes (lane 3). The fragment corresponding to the sequence of the pPROEX HTb plasmid is indicated (5758 pb) (lane 2). C,D) Recombinant NS1 protein was identified by Western blot using two diferentes antibodies anti-NS1 (policlonal antibody) and anti-His (policlonal antibody). The recombinat NS1 protein rNS1 (55 kDa) was detected in the soluble (lane 1) and insoluble (lane 2) fractions but it was absent in bacteria transformed with pPROEX HTb without the sequence of NS1(lane 3).
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Figure S2. NS1-His purification by affinity chromatography
Figure S2. NS1-His purification by affinity chromatography. A) Recombinant NS1 protein from supernatants of IPTG induced bacteria was purified by metal affinity resin (TALON, Clontech). The column was washed three times with buffer A and five times with buffer A in the presence of different concentrations of imidazole. The purified rNS1 protein was maintained bound to the resin (R). Proteins present in each fraction were analyzed by 8% SDS-PAGE and stained with Coomassie blue. B) Western blot analysis of purified rNS1 using anti-NS1 antibody. The band of 55 kDa, enriched in the R fraction, was detected by the anti-NS1 antibody. C) Western blot analysis of the rNS1 run in a native gel, using anti-NS1 antibody from the soluble (lane 1) and insoluble (lane 2) fractions of bacteria transformed with pPROEX HTb-NS1 (lanes 1 and 2) o with pPROEX HTb without NS1 (lane 3).
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Figure S3. Negative control obtained by affinity chromatography
Figure S3. Negative control obtained by affinity chromatography. Total extract from IPTG induced bacteria transformed with pPREOEX HTb plasmid without NS1 sequence was incubated with a metal affinity resin (TALON, Clontech). The column was washed three times with buffer A and five times with buffer A in the presence of different concentrations of imidazole. Proteins present in each fraction were analyzed by 8% SDS-PAGE and stained with Coomassie blue. B) Western blot analysis of purified proteins using anti-NS1 antibody. No bands were detected in the resin (R fraction) and no proteins were detected with the anti-NS1 antibody.
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Figure S4. Purification of cell proteins that interact with NS1 by affinity chromatography. Cytoplasmic extracts of Huh-7 cells were incubated with 500 µl of resin (TALON, Clontech) coupled to rNS1 (A) or with the negative control resin (B) overnight at room temperature. Resins were washed six times with different concentrations of imidazole and the NS1-interacting proteins were eluted with 1 and 1.5 M of NaCl. Proteins present in each fraction were analyzed by 10% SDS-PAGE and stained with Coomassie blue. Proteins present in the elution fraction (1M of NaCl) were identified by Maldi-ToF . * marks the elution fraction which was used for proteins identification.
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Figure S5. Immunoprecipitation of NS1 from infected cells extracts
Figure S5. Immunoprecipitation of NS1 from infected cells extracts. A) 50 ul of protein G-agarose were cross-linking with NS1 antibody, beads were interacted overnight with clarified Huh-7 cytoplasmic extract (2mg/ml) at room temperature. After 5 washes with washing buffer the immunoprecipitated proteins were eluted from the beads and analyzed by SDS-PAGE gel for subsequent identification by mass spectrometry (Maldi-Tof). B) We used as a negative control antibody unrelated rabbit IgG which was subjected to the same working conditions. * marks the elution fraction which was used for proteins identification. E) elution P) protein G-agarose after all washed.
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Figure S6. NS1 interacts with GAPDH, RPL18a and RPL18 proteins
Figure S6. NS1 interacts with GAPDH, RPL18a and RPL18 proteins. Infected cells extracts from Huh-7 cells, treated with micrococcal nuclease, were immunoprecipitated with anti-NS1 (panels A and C), anti-GAPDH (panel A) or anti-RPL18 and anti-RPL18a antibodies (panel B and C). The immunoprecipitated proteins (panels A and C) or the total proteins from cytoplasmic extract (panels B and D) were separated by SDS-PAGE and analyzed by Western blot assay using anti-NS1 (panels A-D), anti-GAPDH (panel B) and anti-RPL18 and anti-RPL18a antibodies (panel B and C).
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Figure S7. RPL18 silencing does not inhibit cellular translation
Figure S7. RPL18 silencing does not inhibit cellular translation. Huh-7 cells were transfected with 150 nM of RPL18 (lanes 1 and 3) or with an unrelated siRNA (lanes 2 and 4). Methionine S35 incorporation into cellular proteins were performed after 48 (lanes 1 and 2) and 72 hrs (lane 3 and 4) post transfection. Coomasie blue staining of the gel is presented (A). Total cell proteins from Huh-7 cells transfected with 150 nM of RPL18 (lanes 1 and 3) or with an unrelated siRNA (lanes 2 and 4) were analyzed by SDS-PAGE and silver stained (B). Results are representatives of three independent experiments.
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