sorcin sirna  (Santa Cruz Biotechnology)

Journal: Journal of Molecular Endocrinology

Article Title: Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

doi: 10.1530/jme-17-0153

Figure Lengend Snippet: Figure 1: Expression of sorcin during the period of early pregnancy and during estrous cycle in mouse

Article Snippet: 128 129 2.4 In vivo sorcin knock-down 130 Pregnant female mice underwent mini laparotomy under anesthesia on D3 of pregnancy to 131 deliver 50 nM of sorcin siRNA (sc-41017; Santa Cruz, CA) in a final volume of 25 μl with 132 Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s 133 instructions in one of the uterine horn.

Techniques: Expressing

Journal: Journal of Molecular Endocrinology

Article Title: Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

doi: 10.1530/jme-17-0153

Figure Lengend Snippet: Figure 2: Immunohistochemistry of sorcin expression in mouse uteri during early pregnancy. Sorcin expression was measured in endometrium i.e. glandular epithelium (GE), luminal epithelium (LE), and stroma (S) at different days of pregnancy (D1, D4, D5, D6 and D7). Graph in lower panel shows image score analysis. IS, implantation site; NP, non-pregnant; NC, Negative control. p values are ap<0.001 vs.

Article Snippet: 128 129 2.4 In vivo sorcin knock-down 130 Pregnant female mice underwent mini laparotomy under anesthesia on D3 of pregnancy to 131 deliver 50 nM of sorcin siRNA (sc-41017; Santa Cruz, CA) in a final volume of 25 μl with 132 Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s 133 instructions in one of the uterine horn.

Techniques: Immunohistochemistry, Expressing, Negative Control

Journal: Journal of Molecular Endocrinology

Article Title: Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

doi: 10.1530/jme-17-0153

Figure Lengend Snippet: Figure 3: Effect of sorcin inhibition on the number of implantation sites. The right horn was injected with

Article Snippet: 128 129 2.4 In vivo sorcin knock-down 130 Pregnant female mice underwent mini laparotomy under anesthesia on D3 of pregnancy to 131 deliver 50 nM of sorcin siRNA (sc-41017; Santa Cruz, CA) in a final volume of 25 μl with 132 Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s 133 instructions in one of the uterine horn.

Techniques: Inhibition, Injection

Journal: Journal of Molecular Endocrinology

Article Title: Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

doi: 10.1530/jme-17-0153

Figure Lengend Snippet: Figure 5: Role of ovarian hormones in regulation of sorcin expression. (A) Ovariectomized mice treated with

Article Snippet: 128 129 2.4 In vivo sorcin knock-down 130 Pregnant female mice underwent mini laparotomy under anesthesia on D3 of pregnancy to 131 deliver 50 nM of sorcin siRNA (sc-41017; Santa Cruz, CA) in a final volume of 25 μl with 132 Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s 133 instructions in one of the uterine horn.

Techniques: Expressing

Journal: Journal of Molecular Endocrinology

Article Title: Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

doi: 10.1530/jme-17-0153

Figure Lengend Snippet: Figure 6: Effect of sorcin silencing on cytosolic free Ca+2 , VEGF level, VEGFR-2 expression in mouse

Article Snippet: 128 129 2.4 In vivo sorcin knock-down 130 Pregnant female mice underwent mini laparotomy under anesthesia on D3 of pregnancy to 131 deliver 50 nM of sorcin siRNA (sc-41017; Santa Cruz, CA) in a final volume of 25 μl with 132 Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s 133 instructions in one of the uterine horn.

Techniques: Expressing

Journal: Journal of Molecular Endocrinology

Article Title: Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

doi: 10.1530/jme-17-0153

Figure Lengend Snippet: Figure 7: The proliferation, migration and invasion assay of HUVEC cells. (A) The proliferation of HUVECs was detected in various groups; HUVECs were cultured with supernatants (20% V/V) from mouse primary EECs transfected with sorcin siRNA or scrambled siRNA. In additional group, a blocking anti-VEGF antibody was added in the supernatant from scrambled siRNA treated EECs. Details have been given in “materials and

Article Snippet: 128 129 2.4 In vivo sorcin knock-down 130 Pregnant female mice underwent mini laparotomy under anesthesia on D3 of pregnancy to 131 deliver 50 nM of sorcin siRNA (sc-41017; Santa Cruz, CA) in a final volume of 25 μl with 132 Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s 133 instructions in one of the uterine horn.

Techniques: Migration, Invasion Assay, Cell Culture, Transfection, Blocking Assay

Journal: Journal of Molecular Endocrinology

Article Title: Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

doi: 10.1530/jme-17-0153

Figure Lengend Snippet: Figure 8: Schematic hypothetical representation of the molecular mechanism of sorcin in endometrium

Article Snippet: 128 129 2.4 In vivo sorcin knock-down 130 Pregnant female mice underwent mini laparotomy under anesthesia on D3 of pregnancy to 131 deliver 50 nM of sorcin siRNA (sc-41017; Santa Cruz, CA) in a final volume of 25 μl with 132 Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific) per manufacturer’s 133 instructions in one of the uterine horn.

Techniques:

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Sequestration of sorcin by aberrant forms of tau results in the defective calcium homeostasis

doi: 10.4196/kjpp.2016.20.4.387

Figure Lengend Snippet: HEK293 cells were transiently transfected with T4 (A, B) or T4C3 (C, D) constructs in the absence or presence of sorcin overexpression or sorcin siRNA. After 48 hrs of transfection, cells were treated 2.5 µM thapsigartin. Cell viability was measured with MTT assay (A, C) and statistical analyses of cell viability were determined at 48 hr after thapsigartin treatment (B, D). Data were obtained from independent experiments (n=3) and expressed as mean±SD. * p<0.05 and ** p<0.01 indicate significant differences between the indicated groups. Vec stands for vector (pcDNA3.1-), Sor for sorcin, Cont for control, and Tg for thapsigargin.

Article Snippet: Sorcin siRNA and control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Construct, Over Expression, MTT Assay, Plasmid Preparation, Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Sequestration of sorcin by aberrant forms of tau results in the defective calcium homeostasis

doi: 10.4196/kjpp.2016.20.4.387

Figure Lengend Snippet: Representative immunoblots (A) and quantitative analyses of cleaved PARP (B) and cleaved caspase-12 (C). HEK293 cells were transiently transfected T4 or T4C3 constructs and then co-transfected with sorcin siRNA or control siRNA, respectively. After 48 hr of transfection, cells were treated with 2.5 µM thapsigartin for 24 hr. Western blotting was carried out with cleaved PARP and cleaved caspase-12 antibodies to determine role of sorcin on thapsigartin-induced apoptosis. Data were obtained from independent experiments (n=3) and expressed as mean±SD. ** p<0.01 indicates significant differences between the indicated groups. Vec stands for vector Sor for sorcin, Cont for control, and Tg for thapsigargin.

Article Snippet: Sorcin siRNA and control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection, Construct, Control, Plasmid Preparation

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: (A) Identification of sorcin in microarray. Ab, antibody. (B) Sorcin interacts with NS5A protein. HEK293T cells were transiently transfected with a Myc-tagged NS5A plasmid. Total cell lysates were harvested and incubated with either purified GST or GST-tagged sorcin. GST-bound protein was detected by immunoblot analysis using an anti-Myc monoclonal antibody. (C) HEK293T cells were cotransfected with Myc-tagged NS5A and V5-tagged sorcin expression plasmids. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Myc monoclonal antibody, and then bound proteins were detected by immunoblot assay using an anti-V5 monoclonal antibody. Protein expression of Myc-tagged NS5A and V5-tagged sorcin was verified by immunoblotting with the indicated antibody. (D) NS5A protein interacts with the endogenous sorcin in HCV replicating cells. Huh7.5 cells were electroporated with 10 μg of Jc1 RNA. Cells lysates harvested at 4 days after electroporation were immunoprecipitated with either control serum or an anti-NS5A antibody. Bound protein was immunoblotted with an anti-sorcin antibody. Immunoprecipitation efficiency was verified by immunoblot analysis using an anti-NS5A antibody (lower panel). (E) Huh7.5 cells were either mock infected or infected with Jc1 for 4 h. At 48 h postinfection, cells were transfected with V5-tagged sorcin. At 24 h after transfection, cells were fixed in 4% paraformaldehyde, and immunofluorescence staining was performed by using an anti-V5 monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG to detect V5-tagged sorcin (green); a rabbit anti-NS5A antibody and TRITC-conjugated donkey anti-rabbit IgG were used to detect NS5A (red). Dual staining showed colocalization of sorcin and NS5A as yellow fluorescence in the merged image. Cells were counterstained with DAPI to label nuclei (blue). (F) Huh7.5 cells were either mock infected or infected with Jc1 for 4 h. At 48 h postinfection, cells were fixed in 4% paraformaldehyde, and immunofluorescence staining was performed by using an anti-sorcin monoclonal antibody and TRITC-conjugated donkey anti-mouse IgG to detect endogenous sorcin (red); a rabbit anti-NS5A antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG were used to detect NS5A (green). Dual staining showed colocalization of endogenous sorcin and NS5A as yellow fluorescence in the “Crop” image. IP, immunoprecipitation; IB, immunoblot; M1(R/G), Mander's colocalization coefficient (ratio of red and green fluorophore colocalization).

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Microarray, Transfection, Plasmid Preparation, Incubation, Purification, Western Blot, Expressing, Immunoprecipitation, Electroporation, Control, Infection, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: HCV NS5A interacts with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin is involved in protein interaction. (A) Schematic illustration of both the wild type (WT) and mutants of HCV NS5A. (B) HEK293T cells were cotransfected with the indicated combinations of expression plasmids. At 48 h after transfection, cell lysates were immunoprecipitated (IP) with an anti-V5 monoclonal antibody, and bound protein was detected by immunoblot (IB) analysis with an anti-Myc antibody. (C) Schematic illustration of both the wild type and sorcin mutants. (D) HEK293T cells were cotransfected with Myc-tagged NS5A and either the wild type or mutants of V5-tagged sorcin expression plasmids. Cell lysates harvested at 48 h after transfection were immunoprecipitated with an anti-Myc monoclonal antibody, and bound protein was detected by immunoblot analysis using an anti-V5 antibody. aa, amino acids.

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Phospho-proteomics, Residue, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: Sorcin is phosphorylated at threonine residue 155 by PLK1 in the presence of NS5A. (A) HEK293T cells were cotransfected with Myc-tagged NS5A and V5-tagged wild-type and mutant constructs of sorcin. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-V5 monoclonal antibody and then immunoblotted with an anti-phospho-threonine (p-Thr) monoclonal antibody. Bound proteins were also immunoblotted with an anti-Myc monoclonal antibody. (B) Huh7.5 cells were transfected with either 20 nM negative siRNA or a PLK1-specific siRNA. At 24 h after transfection, cells were further transfected with V5-tagged sorcin in the absence or presence of Myc-tagged NS5A plasmid. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-V5 monoclonal antibody, and bound proteins were immunoblotted with an anti-Myc monoclonal antibody. Knockdown efficiency of PLK1 was verified by immunoblot assay using an anti-PLK1 monoclonal antibody. Phosphorylation of sorcin was analyzed by immunoblotting with an anti-phospho-threonine monoclonal antibody using the same immunoprecipitates. (C) Huh7.5 cells were either mock infected or infected with Jc1 for 4 h and then further transfected with V5-tagged sorcin. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-V5 monoclonal antibody, and bound proteins were immunoblotted with the indicated antibodies. Phosphorylation of sorcin was analyzed by immunoblotting with an anti-phospho-threonine monoclonal antibody using the same immunoprecipitates. (D) Huh 7.5 cells were infected with Jc1 for 4 h and then harvested at the indicated time points. Cell lysates were immunoprecipitated with an anti-sorcin antibody, and bound protein was immunoblotted with an anti-NS5A antibody. Phosphorylation of sorcin was analyzed by immunoblotting with an anti-phospho-threonine monoclonal antibody. p.i., postinfection. (E) Huh7.5 cells were cotransfected with V5-tagged sorcin in the absence or presence of Myc-tagged NS5A plasmid. Cells were either left untreated or treated with 1 μM BI2536 PLK1 inhibitor. At 24 h after treatment, cell lysates were immunoprecipitated with an anti-V5 monoclonal antibody, and bound proteins were immunoblotted with an anti-Myc monoclonal antibody. Expression of sorcin was verified by immunoblotting with an anti-V5 antibody. Phosphorylation of sorcin was analyzed by immunoblotting with an anti-phospho-threonine monoclonal antibody. (F) HEK293T cells were cotransfected with V5-tagged sorcin, Flag-tagged PLK1, and Myc-tagged NS5A. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-V5 antibody, and bound proteins were immunoblotted with either an anti-Flag or an anti-Myc antibody. Protein expression was verified by immunoblotting with the indicated antibodies.

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Residue, Mutagenesis, Construct, Transfection, Immunoprecipitation, Plasmid Preparation, Knockdown, Western Blot, Phospho-proteomics, Infection, Expressing

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: Sorcin is required for HCV propagation. (A) Huh7.5 cells were transfected with a 20 nM concentration of the indicated siRNAs. At 2 days after siRNA transfection, cells were infected with Jc1. At 48 h postinfection both RNA and protein levels were analyzed by qRT-PCR and immunoblot assay, respectively. (B) Naive Huh7.5 cells were infected with Jc1 harvested from cultured supernatants of the experiment shown in panel A. At 48 h postinfection, both HCV RNA and protein levels were determined. Results are presented as a percentage of negative siRNA (means ± SDs; n = 3). The asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001) from the value for the negative control. (C) Huh7.5 cells were transfected with a 20 nM concentration of the indicated siRNAs. At 2 days after transfection, cells were infected with green fluorescent protein-tagged Jc1. At 48 h postinfection, HCV infectivity was determined by limiting dilution assays. Infected cells were assessed by fluorescence microscope. TCID50, 50% tissue culture infectious dose. (D) Huh7.5 cells were transfected with a 20 nM concentration of the indicated siRNAs, and cell viability was assessed by MTT assay. (E) Huh7.5 cells were transfected with the indicated siRNAs. At 24 h after siRNA transfection, cells were further transfected with either a wild-type or siRNA-resistant sorcin mutant plasmid, followed by Jc1 infection. At 48 h postinfection, cell lysates were immunoblotted using the indicated antibodies. Positive (or Pos), HCV-specific siRNA targeting the 5′ nontranslated region (NTR) of Jc1; negative (or Neg), the universal negative-control siRNA; V5-sorcin WT, V5-tagged sorcin wild type; V5-sorcin-Res, V5-tagged siRNA-resistant sorcin mutant.

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Transfection, Concentration Assay, Infection, Quantitative RT-PCR, Western Blot, Cell Culture, Negative Control, Fluorescence, Microscopy, MTT Assay, Mutagenesis, Plasmid Preparation

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: Sorcin is not involved in the replication and translation steps of the HCV life cycle. (A) A schematic diagram of the pRL-HL plasmid is shown at top. Huh7.5 cells were transiently transfected with increasing amounts of V5-tagged sorcin expression plasmid together with pRL-HL dual luciferase and a pCH110 β-galactosidase plasmid. At 48 h after transfection, relative luciferase activity was determined (bottom). BGH-pA denotes bovine growth hormone polyadenylation signal sequence. (B) Huh7.5 cells harboring an HCV subgenomic replicon derived from genotype 1b were transfected with the indicated siRNAs. At 72 h after siRNA transfection, both RNA (left) and protein (right) levels were analyzed by qRT-PCR and immunoblot assay, respectively. (C) Huh6 cells harboring an HCV subgenomic replicon derived from genotype 2a were treated as described for panel B, and both RNA (left) and protein (right) levels were determined. Positive, HCV-specific siRNA targeting the 5′ nontranslated region (NTR) of Jc1; negative (or Neg), universal negative-control siRNA.

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Plasmid Preparation, Transfection, Expressing, Luciferase, Activity Assay, Sequencing, Derivative Assay, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: Sorcin is required for a late step of the HCV life cycle. (A and B) Huh7.5 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were transfected with a 20 nM concentration of the indicated siRNAs. At 24 h after siRNA transfection, medium was replaced with fresh DMEM containing antibiotics. At the indicated time points, intracellular RNA levels (A) and extracellular HCV RNA levels (B) were determined by qRT-PCR. (C) Naive Huh7.5 cells were infected with Jc1 harvested from culture supernatants of the experiment shown in panel A, and viral infectivity was determined by measuring intracellular HCV RNA levels by qRT-PCR. (D) Huh7.5 cells treated as described in panel A were lysed with three cycles of freezing and thawing and centrifuged at 15,000 × g for 15 min in a 4°C microcentrifuge. The supernatant was collected to determine intracellular HCV infectivity. Naive Huh7.5 cells were infected with Jc1 harvested from the intracellular supernatant for 4 h. At 48 h postinfection, relative intracellular HCV infectivity was determined by qRT-PCR. (E) Huh7.5 cells were infected with Jc1 for 4 h (upper panel). At 48 h postinfection, cells were transfected with a 20 nM concentration of the indicated siRNAs. At the indicated time points, protein levels were determined by immunoblot assays using the indicated antibodies. For the second infection (lower panel), naive Huh7.5 cells were infected with Jc1 harvested from culture supernatants of the experiment described for the upper panel. At 48 h postinfection, protein levels were determined by immunoblot assays using the indicated antibodies. Negative (or Neg), universal negative-control siRNA.

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Infection, Transfection, Concentration Assay, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: Sorcin is involved in the assembly step of the HCV life cycle. (A) Huh7.5 cells were infected with Jc1 for 4 h. At 48 h postinfection, cells were transfected with a 20 nM concentration of either a negative or sorcin-specific siRNA. At 48 h after transfection, cells were either left untreated (lanes 1, 2, 4, and 5), treated with 0.5 μg/ml proteinase K (lanes 2 and 5) for 1 h on ice, or treated with 5% Triton X-100 prior to proteinase K treatment (lanes 3 and 6). The amounts of protease-resistant core protein were determined by immunoblot assay. (B) Huh7.5 cells were infected with Jc1 and transfected with a 20 nM concentration of either negative or sorcin-specific siRNA as described for panel A. At 24 h after siRNA transfection, cells were transfected with vector, an siRNA-resistant sorcin mutant (sorcin-Res), and a phosphorylation-defective siRNA-resistant sorcin mutant (sorcin-Res+T155A) expression plasmid. At 48 h after transfection, samples were immunoblotted with the indicated antibodies. Negative, universal negative-control siRNA.

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Infection, Transfection, Concentration Assay, Western Blot, Plasmid Preparation, Mutagenesis, Phospho-proteomics, Expressing, Negative Control

Journal: Journal of Virology

Article Title: Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

doi: 10.1128/JVI.02493-15

Figure Lengend Snippet: Calcium-binding activity of sorcin is required for HCV propagation. (A) HEK293T cells were cotransfected with a Myc-tagged NS5A and V5-tagged wild-type or mutant sorcin plasmid. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Myc monoclonal antibody, and bound proteins were immunoblotted with an anti-V5 antibody. Immunoprecipitation efficiency was verified by immunoblotting with an anti-Myc monoclonal antibody using the same lysates. (B) Huh7.5 cells were transfected with the indicated siRNAs. At 24 h after siRNA transfection, cells were transfected with the indicated combinations of expression plasmids and then infected with Jc1 for 4 h. At 48 h postinfection, cell lysates were immunoblotted using the indicated antibodies. Band intensities of HCV NS5A protein were normalized against those of β-actin. Pos, HCV-specific siRNA targeting the 5′ nontranslated region (NTR) of Jc1; Neg, universal negative-control siRNA; V5-sorcin-Res, V5-tagged siRNA-resistant sorcin mutant; V5-sorcin-Res+F112L, V5-tagged siRNA-resistant calcium-binding-defective sorcin mutant.

Article Snippet: siRNAs targeting sorcin (sorcin siRNA 1, 5′-CUC AGG AUC CGC UGU AUG G-3′ [sense] and 5′-CCA UAC AGC GGA UCC UGA G-3′ [antisense]; sorcin siRNA 2, 5′-AAU GCU GGA UAG AGA UAU GUC-3′ [sense] and 5′-GAC AUA UCU CUA UCC AGC AUU-3′ [antisense]) and the universal negative-control siRNA were purchased from Bioneer (South Korea).

Techniques: Binding Assay, Activity Assay, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Expressing, Infection, Negative Control