Journal: Experimental & Molecular Medicine

Article Title: Flightless-1 inhibits ER stress-induced apoptosis in colorectal cancer cells by regulating Ca 2+ homeostasis

doi: 10.1038/s12276-020-0448-3

Figure Lengend Snippet: a Sorcin expression levels were analyzed by western blot and quantitative real-time PCR. Data are shown as the mean ± SEM ( n = 3). *** p < 0.001, shRNA-Ctrl cells vs. shRNA-FliI cells. b After cotransfection of vector or sorcin with D1ER, FRET signals were measured. Data are shown as the mean ± SEM. *** p < 0.001, shRNA-Ctrl cells vs. shRNA-FliI cells. c Ctrl- and FliI-KD cells were treated with 50 nM TG for 6 h following expression of vector or sorcin. Cell lysates were analyzed by western blotting for phospho-PERK, phospho-IREα, GRP78/BiP, CHOP, sorcin, FliI, and tubulin.

Article Snippet: Mouse sorcin expression plasmids were purchased from Sino Biological (BDA, Beijing, China).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, shRNA, Cotransfection, Plasmid Preparation

Journal: Diabetes

Article Title: Glucose-Induced Nuclear Shuttling of ChREBP Is Mediated by Sorcin and Ca 2+ Ions in Pancreatic β-Cells

doi: 10.2337/db10-1329

Figure Lengend Snippet: Sorcin silencing decreases glucose-stimulated insulin secretion in MIN6 β-cells. MIN6 β-cells were transfected with sorcin-specific or scrambled siRNAs before insulin secretion assay as described in . Data are means ± SEM from 3 separate experiments.

Article Snippet: To assess the effect of sorcin overexpression or silencing on endogenous ChREBP nuclear localization, dissociated islets were transduced with either sorcin (also expressing GFP) or GFP adenoviral particles as above or with shRNA sorcin or control lentiviral particles (Santa Cruz) according to the manufacturer’s instructions in 11 mmol/L glucose RPMI 1640 for 48 h followed by 1 h in 3 mmol/L glucose RPMI 1640 and 6 h in 17 mmol/L glucose RPMI 1640.

Techniques: Transfection

Journal: Diabetes

Article Title: Glucose-Induced Nuclear Shuttling of ChREBP Is Mediated by Sorcin and Ca 2+ Ions in Pancreatic β-Cells

doi: 10.2337/db10-1329

Figure Lengend Snippet: ChREBP-EGFP nuclear translocation evoked by [Ca 2+ ] cyt requires sorcin for coupling of the events. A - F : Live-cell images of ChREBP-EGFP in MIN6 β-cells (typical of n ≥7–10 cells) expressed either alone or in combination with sorcin siRNA ( F ; n >10). All cells starved (3 mmol/L glucose) overnight and were imaged while perifusing with indicated buffers in warm KRBH. In C , cells were preincubated (10 min) with nifedipine (nif; 10 μmol/L) and diazoxide (diaz; 250 μmol/L) before imaging. Unless specified as Ca 2+ -free (containing EGTA), all buffers contained 1.5 mmol/L Ca 2+ . Scale bar = 10 μm. G - I (upper panels): [Ca 2+ ] cyt signals evoked by glucose ( G , I ) and ATP ( H , I ). Traces show the responses from ≥30 cells (≥55 for sorcin-silenced cells), taken from at least 3 independent experiments (means ± SEM). G - I (bottom panels): Real-time quantification of ChREBP-EGFP (gray values) in nuclear and cytosolic compartments. Traces (scale reversed to time-match events in the upper panel) show average from ≥10 cells ( I ; ≥30) during various treatments, from 3 independent experiments (means ± SEM). nuc, nucleus; cyto, cytosol; glu, glucose. ** P < 0.005, *** P < 0.0005 for the effects of treatments. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: To assess the effect of sorcin overexpression or silencing on endogenous ChREBP nuclear localization, dissociated islets were transduced with either sorcin (also expressing GFP) or GFP adenoviral particles as above or with shRNA sorcin or control lentiviral particles (Santa Cruz) according to the manufacturer’s instructions in 11 mmol/L glucose RPMI 1640 for 48 h followed by 1 h in 3 mmol/L glucose RPMI 1640 and 6 h in 17 mmol/L glucose RPMI 1640.

Techniques: Translocation Assay, Imaging

Journal: Diabetes

Article Title: Glucose-Induced Nuclear Shuttling of ChREBP Is Mediated by Sorcin and Ca 2+ Ions in Pancreatic β-Cells

doi: 10.2337/db10-1329

Figure Lengend Snippet: Sorcin colocalizes with ChREBP in MIN6 β-cells and controls its subcellular localization. A and B : MIN6 β-cells were transfected with c- myc –ChREBP alone ( A ) or in combination with sorcin-HA ( B ) and cultured in either 3 or 30 mmol/L glucose for 6 h as indicated. Immunocytochemistry and confocal imaging were performed as described in . Scale bars = 5 μm. C : Quantification of c- myc –ChREBP in the nucleus, for the 30 mmol/L glucose experiments presented in A and B . The intensities of c- myc staining were measured using Volocity 4.0 software, for both nuclear and cytoplasmic regions of interest, in each individual cell, and background intensities were subtracted. Nuclear ChREBP intensity is expressed as percentage of cytoplasmic intensity ( n ≥ 3 experiments, with 30 cells per condition). D : MIN6 β-cells were transfected with sorcin-HA in 25 mmol/L Dulbecco’s modified Eagle’s medium for 48 h before culturing in 3 mmol/L glucose for 16 h, followed by 3 or 30 mmol/L glucose for 6 h. The nuclear and cytosolic fractions were extracted, and Western blots were performed as described in . The blots were probed with anti-ChREBP ( top ), anti-HA ( middle ), or anti–α-tubulin antibodies ( bottom ). The experiment was repeated twice with similar results. E : MIN6 β-cells were cotransfected with either −148L-PK luci, −183L-PK luci, or −1081TXNIP luci together with GFP or sorcin as indicated before cell lysis and luciferase assays as described in ( n = 3 to 8). GFP, green fluorescent protein. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: To assess the effect of sorcin overexpression or silencing on endogenous ChREBP nuclear localization, dissociated islets were transduced with either sorcin (also expressing GFP) or GFP adenoviral particles as above or with shRNA sorcin or control lentiviral particles (Santa Cruz) according to the manufacturer’s instructions in 11 mmol/L glucose RPMI 1640 for 48 h followed by 1 h in 3 mmol/L glucose RPMI 1640 and 6 h in 17 mmol/L glucose RPMI 1640.

Techniques: Transfection, Cell Culture, Immunocytochemistry, Imaging, Staining, Software, Modification, Western Blot, Lysis, Luciferase

Journal: Diabetes

Article Title: Glucose-Induced Nuclear Shuttling of ChREBP Is Mediated by Sorcin and Ca 2+ Ions in Pancreatic β-Cells

doi: 10.2337/db10-1329

Figure Lengend Snippet: Sorcin controls ChREBP subcellular location in primary mouse β-cells. A : Dissociated mouse islets were transduced for 48 h with null or sorcin-overexpressing adenoviruses in 11 mmol/L glucose RPMI 1640, and then incubated 1 h in 3 mmol/L glucose followed by 6 h in 17 mmol/L RPMI 1640, before immunochemistry and confocal imaging as described in . Scale bars = 5 μm. C : Dissociated mouse islets were transduced with control or sorcin-shRNA lentiviruses for 72 h in 11 mmol/L RPMI 1640 and then incubated for 6 h in 3 mmol/L RPMI 1640 before immunochemistry and confocal imaging as above. B and C : Quantification of endogenous ChREBP in the nucleus, for the experiments presented in A and C . The intensities of ChREBP staining were measured using Volocity 4.0 software, for both nuclear and cytoplasmic regions of interest, in each individual cell, and background intensities were subtracted. Nuclear ChREBP intensity is expressed as percentage of cytoplasmic intensity ( n = 3 experiments, with 24 to 29 cells per condition). (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: To assess the effect of sorcin overexpression or silencing on endogenous ChREBP nuclear localization, dissociated islets were transduced with either sorcin (also expressing GFP) or GFP adenoviral particles as above or with shRNA sorcin or control lentiviral particles (Santa Cruz) according to the manufacturer’s instructions in 11 mmol/L glucose RPMI 1640 for 48 h followed by 1 h in 3 mmol/L glucose RPMI 1640 and 6 h in 17 mmol/L glucose RPMI 1640.

Techniques: Incubation, Imaging, Transduction, Control, shRNA, Staining, Software

Journal: Diabetes

Article Title: Glucose-Induced Nuclear Shuttling of ChREBP Is Mediated by Sorcin and Ca 2+ Ions in Pancreatic β-Cells

doi: 10.2337/db10-1329

Figure Lengend Snippet: Proposed model of ChREBP activation via sorcin and Ca 2+ ions. High glucose and subsequent Ca 2+ -induced Ca 2+ release (CICR) cause ChREBP and sorcin dissociation in the cytosol. Activated ChREBP then translocates into the nucleus where it activates transcription. IP3R, inositol 1,4,5-trisphosphate receptors; ER, endoplasmic reticulum; RyR, ryanodine receptors; PLC, phospholipase C; VGCC, voltage-gated calcium channels; Nuc, nucleus. (A high-quality color representation of this figure is available in the online issue.)

Article Snippet: To assess the effect of sorcin overexpression or silencing on endogenous ChREBP nuclear localization, dissociated islets were transduced with either sorcin (also expressing GFP) or GFP adenoviral particles as above or with shRNA sorcin or control lentiviral particles (Santa Cruz) according to the manufacturer’s instructions in 11 mmol/L glucose RPMI 1640 for 48 h followed by 1 h in 3 mmol/L glucose RPMI 1640 and 6 h in 17 mmol/L glucose RPMI 1640.

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI and STAT3 are upregulated both in HCC tissues and cells. ( A , B ) SRI and STAT3 are highly expressed in HCC tissues (T) compared with normal liver tissues (N) in the GEO database (GDS4882). ( C ) Image available from Proteinatlas database showed high expression of SRI and STAT3 in tumor (T) compared with normal liver tissues (N). The expression of proteins were determined by the brown area. Scale bars = 200 μm. ( D – H ) Western blot showed SRI and STAT3 proteins were overexpressed in clinical HCC tissues and in Huh-7/HepG2/Hep3B cells. The red and blue spots represented the expression of different proteins in the T and N groups, respectively. ( I ) From the TGGA database, SRI was found to be positively correlated with STAT3 in LIHC ( p < 0.001). ( J , K ) The expressions of SRI and STAT3 are positively correlated in HCC tissues and cells lines ( p < 0.05). * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI and STAT3 are interacting proteins. ( A ) SRI and STAT3 are interacting proteins in the STRING database. ( B ) SRI and STAT3 are interacting proteins confirmed by co-immunoprecipitation assays in Huh-7/HepG2 cells. ( C , D ) SRI (shown in red) co-localized with STAT3 (shown in green) was visualized by cellular immunofluorescence in Huh-7/HepG2 cells. DAPI (shown in blue) was used for nuclear staining. Scale bars = 200 μm.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Immunoprecipitation, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI interacts with STAT3, inhibits apoptosis and activates the NF-κB signaling pathway in vitro and in vivo. ( A – D ) SRI downexpression reduced the fluorescence intensity of TMRE (shown in red) and enhanced the apoptosis sensitivities by Hoechst 33342 (shown in blue) staining assay. The results of SRI overexpression were consisted with SRI downexpression. Scale bars = 200 μm. ( E – J ) Western blot detected the expression of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins when SRI overexpression or downexpression were found in HCC cell and tumor xenografts. ( K , L ) Representative IHC images of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins when SRI overexpression or downexpression were found in tumor xenografts. The expression of proteins were determined by the brown area. Scale bars = 50 μm. * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: In Vitro, In Vivo, Fluorescence, Staining, Over Expression, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI and STAT3 interaction is crucial for anti-apoptosis. ( A – D ) Stattic reduced the fluorescence intensity of TMRE (shown in red) and enhanced the sensitivities to SRI induced apoptosis by Hoechst 33342 (shown in blue) staining assay. Scale bars = 200 μm. ( E – H ) The expression of SRI, p65, p-p65, p-IκB and apoptosis-related proteins were detected by Western blot in Stattic group. * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Fluorescence, Staining, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: SRI inhibits cells apoptosis through the NF-κB signaling pathway. ( A , B ) Tumor volume and weight of orthotopic xenograft models derived from upSRI cells treated with Stattic. Symbols of different colors represented the weight of tumors in different groups. ( C , D ) Representative IHC images of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins in Stattic treatment. Scale bars = 50 μm. ( C , E ) Representative IHC images of p65 and apoptosis-related proteins after treatment with AL inhibitor. The expression of proteins were determined by the brown area. ( F – H ) AL inhibits the fluorescence intensity of TMRE (shown in red) and reduced the sensitivities to SRI-induced apoptosis by Hoechst 33342 (shown in blue) staining assay. Scale bars = 200 μm. ( I – L ) Apoptosis-related proteins were detected after treated with AL inhibitor by Western blot. * p < 0.05, ** p < 0.01.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Derivative Assay, Expressing, Fluorescence, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

doi: 10.3390/ijms25137206

Figure Lengend Snippet: A schematic illustration of SRI regulating apoptosis in HCC. SRI overexpression inhibits the apoptosis of HCC cells by interacting with STAT3 via NF-κB pathway, whereas opposing effects were observed for knockdown of SRI. Stattic, an inhibitor of STAT3, promotes apoptosis of HCC cells via NF-κB pathway. Avicularin, the inhibitor of NF-κB, which promotes apoptosis of HCC cells. ↑ : represent upregulation or promotion. ↓ : represent downregulation or inhibition.

Article Snippet: The diluted primary antibodies against SRI (1:500, Proteintech, Wuhan, China), STAT3 (1:500, Proteintech, Wuhan, China), Bcl-XL (1:500, Proteintech, Wuhan, China), MCL-1 (1:500, Proteintech, Wuhan, China), p65 (1:500, Proteintech, Wuhan, China), p-p65 (1:500, Abmart, Shanghai, China), Bcl-2 (1:1000, Abcam, Cambridge, UK), Bax (1:1000, Proteintech, Wuhan, China), caspase3 (1:500, Proteintech, Wuhan, China), cIAP1 (1:500, Proteintech, Wuhan, China), p-IκB (1:500, Proteintech, Wuhan, China), tubulin (1:5000, Proteintech, Wuhan, China) and GAPDH (1:10000, Proteintech, Wuhan, China) were incubated with the membranes at 4 °C overnight.

Techniques: Over Expression, Knockdown, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin activates the plasma membrane Ca 2+ -ATPase (PMCA) activity and prevents the inhibition of the pump by amyloid-β peptide (Aβ1-42) and tau. Purified PMCA (2.5 μg) was reconstituted with phosphatidylcholine (PC) (○) or phosphatidylserine (PS) (Δ), and incubated for 2 min at 37 °C, with Ca 2+ and increasing concentrations of sorcin, in the absence or presence of 30 µM Aβ1-42 (●■) or 300 nM tau (▲) in 25 µL. Afterward, samples were further diluted up to 1 mL in assay medium (final concentrations of Aβ and tau were 0.75 µM and 7.5 nM, respectively). The Ca 2+ -ATPase activity was measured as described in the Methods Section. Data are mean ± SE of eight experiments performed with three preparations.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Membrane, Activity Assay, Inhibition, Purification, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin does not to protect PMCA activity from its inhibition by Aβ and tau in the presence of EGTA. Purified pig brain PMCA (2.5 μg) reconstituted in PC or PS was treated for 2 min at 37 °C, with 2 mM EGTA, in the absence (plain bars) or presence of 1 µM sorcin (stripped bars) and 30 µM Aβ or 300 nM tau (grey bars) in 25 μL. The mixture was further diluted up to 1 mL in the assay medium (without Ca 2+ ) and the reaction was started by addition of 1 mM ATP and 100 µM CaCl 2 . Data represent mean ± SE from three experiments performed in duplicate and with three different preparations.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activity Assay, Inhibition, Purification

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin activates all PMCA ( A ) and SERCA ( B ) isoforms. Twenty µg of membranes from COS cells overexpressing PMCA or SERCA isoforms were treated in the absence (plain bars) or presence of 1 μM sorcin, and 50 µM CaCl 2 (stripped bars) in 25 µL, and then diluted up to 1 mL with the assay medium plus 0.01% saponin. Activity was measured as indicated in the Methods Section after addition of 1 mM ATP. The 100% activities correspond to 0.124 ± 0.003, 0.107 ± 0.004, 0.093 ± 0.006 and 0.102 ± 0.01 µmol.min −1 .mg −1 for hPMCA1b, hPMCA2b, rPMCA3b and hPMCA4b respectively, and to 2.57 ± 0.018, 0.086 ± 0.015 and 0.216 ± 0.01 µmol.min −1 .mg −1 for SERCA1, SERCA2b and SERCA3, respectively. Data are mean ± SE of three experiments performed in triplicate with three preparations. p ≤ 0.001 vs. control.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Sorcin activates Ca 2+ -ATPase activities of overexpressed full hPMCA4b, their truncated variants and SERCA2b isoforms in a concentration-dependent manner and prevents their inhibition by Aβ and tau. Twenty µg of COS cells’ membranes overexpressing full hPMCA4b, or truncated hPMCA4b-L1086* and hPMCA4b-R1052* and SERCA2b isoforms were treated with increasing concentrations of sorcin, without (○) or with 30 μM Aβ1-42 (●) or 300 nM tau (▲), in 25 µL, and then diluted up to 1 mL with the assay medium plus 0.01% saponin, resulting in the indicated final concentrations of sorcin. Activities were measured as indicated in the Methods Section, after addition of 1 mM ATP. The 100% activities correspond to 0.226 ± 0.02, 0.235 ± 0.03, 0.242 ± 0.02 and 0.190 ± 0.003 µmol. min −1 . mg −1 for hPMCA4b, hPMCA-L1086*, hPMCA-L1052* and SERCA2b, respectively. Data represent mean ± SE of three experiments performed with three preparations.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Concentration Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: ( A ) Sorcin prevents the inhibition of PMCA activity by Aβ and/or tau in membranes from human control (HC) and AD (HAD) brain samples. Ten µg of membranes from HC (Braak stages I and II) and from HAD (Braak stages V and VI) were incubated without (plain bars) or with (stripped bars) 1 µM sorcin and 30 μM Aβ1-42 or 300 nM tau (grey bars) in 25 µL, and then diluted up to 1 mL with assay medium plus 0.01% saponin. PMCA activity was assayed after triggering the reaction with 1 mM ATP, as indicated in the Methods Section. Data represent mean ± SE values of three experiments performed with four preparations of each Braak stage. * p ≤ 0.001 vs. MV without sorcin. ( B ) Expression levels of sorcin in human brain samples at increasing Braak stages of AD. Twenty µg of human brain samples (SN1) of Braak stages I, II, III, IV, V and VI, were electrophoresed into 12% SDS-PAGE gel, electro-transferred to PVDF and immuno-stained with the anti-sorcin antibody, as indicated in the Methods Section. The immunoblot is representative of three assays with at least three samples from each stage. Relative expression of sorcin vs. GAPDH is shown as mean ± SE values (arbitrary units). * p ≤ 0.001 vs. Braak stage I.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Inhibition, Activity Assay, Control, Incubation, Expressing, SDS Page, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Protective effects of sorcin on Ca 2+ -ATPase activity ( A , B ), cell viability ( C ), ROS production ( D ) and apoptosis ( E , F ), in human neuroblastoma cells. SH-SY5Y cells were treated without or with 5 μM Aβ1-42 or 10 nM tau in the absence and presence of 1 μM sorcin for 24 h, as detailed in the Methods Section. ( A ) Ca 2+ -ATPase activity was assayed in 10 μg of membranes, previously treated as indicated above. ( B ) Ca 2+ -ATPase activity was measured by incubating non-treated cells with 30 μM Aβ1-42 or 300 nM tau in the absence and presence of 1 µM sorcin, in 25 µL, and then diluted up to 1 mL with assay medium plus 0.01% saponin. Activity data are mean ± SE of three independent experiments. Cells treated with Aβ or tau. ( C ) Cell viability was assayed in control and treated cells after 1 h incubation with 150 µg/mL MTT (white bars) or stained with 0.4% trypan blue (black bars). Data are expressed as percentage of untreated cells, as mean ± SE of five independent experiments. ( D ) The production of ROS was determined by incubating non-treated and treated cells with 40 μM H 2 DCFDA, as indicated in the Methods Section. Data are expressed as mean ± SE of fluorescence intensity of three independent experiments. ( E ) DAPI staining in non-treated and treated cells. Representative fluorescent microscopy images show apoptotic cells with condensed and fragmented nuclei (white arrows). Scale bar: 10 µm. Apoptotic nuclei were quantified with respect to the total number of seeded cells. Values are mean ± SE of ten images per coverslip, obtained from three cell cultures. ( F ) Caspase-3 activity was measured in non-treated and treated cell lysates by detecting cleavage of the caspase substrate for 1 h at 37 °C. Data are represented as a percentage relative to non-treated cells (0.206 ± 0.005 fluorescence intensity/µg protein). Values are mean ± SE of three experiments. * p ≤ 0.001 vs. cells treated with Aβ or tau.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activity Assay, Control, Incubation, Staining, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: Blot overlay assays of the interaction between sorcin and PMCA, Aβ and tau. ( A ) PMCA (0.5 μg) and sorcin (1 μg) were run in 10% SDS gel, transferred to PVDF membranes and incubated sequentially with 0.5 µg PMCA and the a-PMCA antibody 5F10. The panel shows binding of PMCA to sorcin. ( B ) Sorcin (1 ug), Aβ (3 µg) and tau (3 µg) were run in three gradient 10–20% gels, transferred to PVDF and incubated sequentially with 3 µg Aβ or 3 µg tau, and with a-Aβ1-42 or a-tau antibodies, as indicated. The panel shows binding of both Aβ and tau to sorcin. It also shows a Western blot of pure sorcin with the a-sorcin antibody, as control. Immunoblots are representatives from three different assays.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: SDS-Gel, Incubation, Binding Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

doi: 10.3390/ijms22116055

Figure Lengend Snippet: The scheme summarizes the interactions of sorcin with PMCA (red) and SERCA (blue), in the presence of Ca 2+ that lead to Ca 2+ -ATPase activation. Sorcin also interacts with AD markers, such as tau and Aβ, preventing their binding to PMCA and their inhibitory effects on PMCA activity.

Article Snippet: Recombinant Human SRI (sorcin) was obtained from Sino Biological US Inc, Eschborn, Germany.

Techniques: Activation Assay, Binding Assay, Activity Assay

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