Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A, hnRNP A2, hnRNP A3, and hnRNP U are part of the same multiprotein complex. (A) Specificity of the affinity-purified peptide specific polyclonal anti-CBF-A antibody. Total protein extracts from HeLa cells were resolved by SDS-PAGE, blotted, and stained with Coomassie Blue (lane 1), immunostained with the CBF-A preimmune serum (lane 2), or with the affinity-purified anti-CBF-A antibody (lane 3) and with antibody SAK22 recognizing both CBF-A isoforms p37 and p42 (lane 4). (B) Sucrose gradient analysis of CBF-A, hnRNP A2, and hnRNP A3 from HeLa nuclear extracts. Fractions were resolved by SDS/PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2/A3. (C) Schematic representation of recombinant hnRNP A2 and A3 and CBF-A constructs. (D) Pulldown experiment using S-tagged hnRNP A2, S-tagged hnRNP A3, or GST-tagged CBF-A constructs. The beads were incubated with HeLa nuclear extracts. Bound proteins were resolved by SDS PAGE, revealed by Coomassie staining and (E) analyzed on immunoblots with antibodies to CBF-A, hnRNP A2 and A3, and hnRNP U.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Affinity Purification, SDS Page, Staining, Western Blot, Recombinant, Construct, Incubation

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A binds the MBP mRNA RTS. (A) Sequences of wild-type (wtRTS) and scrambled RTS (scrRTS) used in this study. (B) Biotinylated wtRTS and scrRTS were conjugated to streptavidin Sepharose. Beads were incubated with HeLa nuclear, cytoplasmic, and high-salt protein extracts. Bound proteins were resolved by SDS-PAGE, revealed with Coomassie, and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2 and A3. (C) RTS-binding assays using 33P-labeled wtRTS and scrRTS sequences. To perform EMSA, wtRTS and scrRTS probes were incubated with purified CBF-A and hnRNP A2 and A3 without affinity tags or (D) in the presence (+) or absence (−) of a 25-fold excess of unlabeled competitor RNA oligonucleotides as indicated. (E) Tissue distribution of CBF-A, analyzed on immunoblots, and normalized to the steady-state expression of histone H3.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Incubation, SDS Page, Western Blot, Binding Assay, Labeling, Purification, Expressing

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In cultured oligodendrocytes, CBF-A exhibits a granular cytoplasmic distribution which correlates with transported MBP mRNA. (A) Endogenous CBF-A (A–D and E–H) or (hnRNP A2 I–L and M–P) and MBP mRNA were simultaneously monitored by immuno-FISH and confocal microscopy. In D, arrows identify sites in which the distribution of CBF-A correlates with MBP RTS along processes. In E–H and M–P, oligodendrocyte processes are shown at approximately fivefold higher magnification. In H, arrowheads identify examples of CBF-A and MBP RTS-positive granules. In P, arrows point to examples of hnRNP A2- and MBP mRNA-positive granules. Scale bar, 20 μm. (B) Unbiased statistical quantification of individual CBF-A and MBP RTS-positive granules and (C) hnRNP A2 and MBP RTS-positive granules based on the immuno-FISH analysis. In both cases a linear correlation between the fluorescence intensity levels of CBF-A and RTS or hnRNP A2 and RTS is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Cell Culture, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: In oli-neu cells, the distribution of endogenous CBF-A correlates with hnRNP A2. (A and E) DAPI staining, (B and F) oli-neu cells stained with a mAb to hnRNP A2. (C and G) Oli-neu cells stained with the rabbit polyclonal peptide-specific anti-CBF-A antibody and (D and H) merged images. Scale bar, 20 μm. (B) Statistical quantification of CBF-A and hnRNP A2-positive granules based on the double immunofluorescence analysis and confocal microscopy in A. A linear correlation between the fluorescence signals of CBF-A and hnRNP A2 is revealed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Staining, Immunofluorescence, Confocal Microscopy, Fluorescence

Journal:

Article Title: In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

doi: 10.1091/mbc.E07-10-1083

Figure Lengend Snippet: CBF-A is associated with MBP mRNA in differentiating oligodendrocytes. (A) A complex containing CBF-A and hnRNP A2 is coprecipitated with the anti-CBF-A antibody from total protein extracts (Input) prepared from differentiating oli-neu cells in an RNA-dependent manner. Where indicated, extracts were treated with RNase A before immunoprecipitation. Bound proteins were resolved by SDS-PAGE and analyzed on immunoblots with antibodies to CBF-A and hnRNP A2. (B) qRT-PCR was performed on reverse-transcribed cDNA derived from RNA extracts of differentiating oli-neu cells, immunoprecipitated by CBF-A. The anti-CBF-A antibody leads to enrichment of MBP mRNA, as assessed with MBP-specific primers. Mock experiments and IgG pulldowns revealed negligible RNA enrichment. Input samples were considered to be 100%; thus all samples were divided by the inputs mean value. Data are presented as average of three independent experiments. Error bars, SEM. Importantly, in each case the percentages of immunoprecipitated mRNA are relative to the total amount of each individual mRNA species (input) analyzed.

Article Snippet: Cloning, Expression, and Protein Purification Full-length hnRNP A2 (forward primer 5′-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5′-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 ( Ma et al. , 2002 ) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Quantitative RT-PCR, Reverse Transcription, Derivative Assay

Journal: Journal of internal medicine

Article Title: Elevated expression of CX3C chemokine receptor 1 mediates recruitment of T cells into bone marrow of patients with acquired aplastic anaemia.

doi: 10.1111/joim.12218

Figure Lengend Snippet: Fig. 5 Chemotaxis and adhesion of T cells. Representative images (a) and quantification of chemotaxis of peripheral blood CD4+ (b) and CD8+ (c) T cells from patients with severe aplastic anaemia (SAA: n = 17) and 10 healthy control subjects (n = 10) in response to autologous bone marrow plasma or soluble fractalkine (CX3CL1) in the absence or presence of the CX3C chemokine receptor 1 antagonist F1 or anti-CX3CL1 neutralizing monoclonal antibodies. Results are expressed as chemotactic index (CI). Representative images (d) and quantification of adhesion of peripheral blood CD4+ (e) and CD8+ (f) T cells in response to immobilized CX3CL1 in the absence or presence of F1. Random adhesion was subtracted, and data were expressed as the mean number of cells per field of view. Data are presented as mean SD. Comparisons between different groups: *P < 0.05, **P < 0.01.

Article Snippet: The levels of CX3CL1 and VCAM-1 were measured using commercially available enzyme-linked immunosorbent assay kits for human CX3CL1 (R&D Systems, Shanghai, China) and human VCAM-1 (Bender MedSystems Gmbh, Vienna, Austria) according to manufacturers’ instructions.

Techniques: Chemotaxis Assay, Control, Clinical Proteomics, Bioprocessing

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: HspBP1 inhibits HIV-1 LTR promoter activity through NF-κB enhancer region. ( A ) Luciferase reporter assays showing inhibition of LTR-driven gene-expression by HspBP1 in dose-dependent manner in HEK293T cells. Cells were co-transfected with increasing amounts of HspBP1 construct (0.25–1.5 ug) and the HIV-1 LTR luciferase reporter construct (LTR-luc) followed by luciferase assay after 36 h of transfection. ( B ) HspBP1 over-expression inhibits LTR promoter activity in dose dependent manner in Jurkat cells. Jurkat cells were co-transfected with 0.5 and 1 ug of HspBP1-construct along with LTR-luc. Luciferase assays were performed 36 h post transfection. ( C ) HspBP1 silencing enhances HIV-1 LTR driven gene expression in dose-dependent manner. Control siRNA and siRNA against HspBP1 were transfected in HEK293T cells. LTR-luc was also transfected 24 h after siRNA-transfection. Luciferase activities were determined 24 h post second transfection. Efficiency of gene silencing was assessed by immunoblotting. ( D ) HspBP1 inhibits LTR-driven gene-expression in presence of Tat. HEK293T cells were co-transfected with indicated plasmids and luciferase assays were performed 36 h post transfection. ( E ) Schematic representation of HIV-1 LTR and LTR-luciferase mutant constructs . ( F ) Luciferase reporter assays depicting inhibition of LTR promoter activity by HspBP1 through NF-κB and SP1 binding sites. HEK293T cells were co-transfected with various LTR luc constructs and HspBP1. Luciferase assays was performed 36 h post-transfection. ( G ) Schematic representation of NF-κB mutants of HIV-1 LTR promoter. ( H ) Effect of HspBP1 on HIV-1 LTR promoter is specifically through NF-κB binding sites. HEK293T cells were co-transfected with indicated plasmids and reporter assays were performed 36 h post-transfection. ( I ) HspBP1 inhibits NF-κB driven gene expression in dose-dependent manner. HEK293T cells were co-transfected with increasing concentrations of HspBP1 vector (0.25–1.5 ug) and luciferase reporter construct under the control of five NF-κB binding sites. Luciferase activities were determined 36 h post-transfection. Results shown in (A), (C), (D) and (I) are representative of three experiments. (B), (F) and (H) represent data from two experiments. Error bars represent the mean ± SD values and significance is defined as * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Activity Assay, Luciferase, Inhibition, Gene Expression, Transfection, Construct, Over Expression, Control, Western Blot, Mutagenesis, Binding Assay, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: List of the primers used for qRT-PCR analysis

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: HspBP1 interacts with both HSP40 and HSP70 and interferes with HIV-1 gene-expression and replication. ( A ) Co-immunoprecipitation showing interaction of HspBP1 with HSP70 and HSP40. HEK293T cells were co-transfected with indicated plasmids. Forty-eight hours post-transfection, immunoprecipitation was performed with indicated antibodies followed by immunoblotting for specified proteins. ( B ) Co-immunoprecipitation showing interaction of HspBP1 with HSP70 and HSP40 at endogenous level in infected CEM-GFP cells. Immunoprecipitation was performed with HspBP1 antibody with day-5 0.1 MOI HIV-1 NL4-3 infected CEM-GFP cells followed by immunoblotting with either HSP40 or HSP70 antibodies. ( C ) Upper panel: p24 ELISA showing the effect of over-expression of different HSPs on virus production. Jurkat cells were transfected with indicated plasmids. Twenty-four hours post-transfection, cells were infected with HIV-1 NL4-3. Twenty-four hours post-infection, cells were harvested and culture supernatants were collected. Culture supernatants were examined for amount of virus produced using p24 antigen capture ELISA. Lower panel: Quantitative real time PCR analysis showing effect of over-expression of different HSPs on viral gene-expression. Cells were utilized to isolate RNA and cDNA was prepared. The expression of p24 mRNA was analyzed using qRT-PCR. ( D ) Luciferase reporter assays showing effect of over-expression of different HSPs on LTR driven gene-expression. HEK293T cells were co-transfected with the indicated plasmids and luciferase assay was performed 36 h post transfection. ( E ) Luciferase reporter assay indicating that the inhibitory function of HspBP1 on viral transcription is independent of HSP40 and HSP70. HEK293T cells were depleted of HSP40 and HSP70 using indicated siRNA. 24 h post siRNA transfection, HIV-1 LTR luciferase reporter construct (LTR-luc) was co-transfected along with indicated plasmids. Luciferase activity was determined 24 h post second transfection. Silencing using siRNA or over-expression of various plasmids was confirmed by immunoblotting in each experiment. (A) and (B) are one of the representative images of two independent experiments. (C), (D) and (E) represent data from three independent experiments. The error bars are presented as the mean ± SD values and significance is defined as * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Gene Expression, Immunoprecipitation, Transfection, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Over Expression, Virus, Produced, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Construct, Activity Assay

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: HspBP1 is down modulated during HIV-1 infection in both T cells and PBMCs. ( A ) mRNA expression profiling of HspBP1 during HIV-1 infection. Jurkat cells were infected with 0.1 MOI HIV-1 NL4-3 and were harvested on day 1 (D1), day 3 (D3), day 5 (D5), day 7 (D7) and day 9 (D9), post infection for quantitative real time PCR analysis. ( B ) Representative western blot showing expression profile of HspBP1 in HIV-1 NL4-3 infected Jurkat cells at different time points. ( C ) Densitometry analysis of HspBP1 expression in western blot shown in (B). ( D ) qRT-PCR analysis of HspBP1 mRNA expression in HIV-1 NL4-3 (0.1MOI) infected PBMCs on day 1 (D1), day 3 (D3) and day 5 (D5) post infection. ( E ) HspBP1 protein expression analysis in HIV-1 NL4-3 infected PBMCs on day 5 (D5) post-infection using immunoblotting. PBMCs were isolated from blood of three healthy donors obtained from local blood bank and were infected with HIV-1 NL4-3 . The expression of viral capsid protein p24 was measured to monitor the progression of infection. The results shown in (A) and (D) are represented as fold change in expression as compared to uninfected cells (UN). The results shown in (A) and (C) are representative of three independent experiments. (D) represents data from two experiments. The error bars are presented as the mean ± SD values and significance is defined as * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Isolation

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: HspBP1 levels inversely correlate with HIV-1 virus production. ( A ) HspBP1 knockdown results in increased HIV-1 virus production in a dose dependent manner in HEK293T cells. Virus production was analyzed by p24 ELISA with culture supernatants from HEK293T cells co-transfected with pNL4-3 and increasing doses of HspBP1 siRNA. Supernatant was collected 48 h post-transfection. ( B ) HspBP1 silencing enhances virus production in HIV-1 infected Jurkat cells. Jurkat cells were transfected with HspBP1 siRNA followed by infection with HIV-1 NL4-3 virus after 24 h. p24 ELISA was performed on the supernatant collected after 48 h of infection. ( C ) HspBP1 over-expression inhibits virus production in dose dependent manner in HEK293T cells. Culture supernatants of HEK293T cells transfected with pNL4-3 and increasing doses of HspBP1 vector were analyzed for virus production using the p24 antigen capture ELISA. ( D ) HIV-1 inhibition by HspBP1 is a specific occurrence as mutant failed to inhibit virus production. HEK293T cells were co-transfected with either wild type or mutant HspBP1 and pNL4-3. Cells were harvested 48 h post-transfection. Virus production was determined by p24 ELISA on culture supernatant. Silencing using siRNA or over-expression of HspBP1 was confirmed by immunoblotting in each experiment. The results are representative of three experiments and presented as the mean ± SD (* P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Virus, Knockdown, Enzyme-linked Immunosorbent Assay, Transfection, Infection, Over Expression, Plasmid Preparation, Inhibition, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: HspBP1 interacts with NF-κB consensus region on LTR promoter. ( A ) Schematic illustration of HIV-1 LTR promoter showing the position of oligonucleotide probe (P) and primers (F1/R1, F2/R2 and F3/R3) used in EMSA and ChIP assays, respectively. ( B ) His-HspBP1 binds to probe in dose-dependent manner. ( C ) His-HspBP1 binding to probe is inhibited by cold excess oligo but not by mutant oligo. ( D ) Probe binds to HEK293T nuclear extract, which is inhibited by pre-incubation with HspBP1 antibody while binding remains unaffected during post-incubation with HspBP1 antibody. ( E ) Binding of probe to nuclear extract is inhibited by HspBP1 antibody in dose-dependent manner while IgG control does not have any effect. ( F ) Specificity of probe binding to nuclear extract is analyzed by competition with specific (cold) and non-specific (mutant) oligos. (G) HspBP1 is recruited on LTR promoter at NF-κB-SP1 enhancer region in HIV-1 NL4-3 infected CEM-GFP cells. Fixed and sonicated chromatin from day 5 infected CEM-GFP cells were immunoprecipitated with HspBP1 antibody followed by qRT-PCR analysis with indicated primers spanning three different regions on LTR promoter. (B), (C), (D), (E) and (F) are one of the representative figure of at least two independent experiments. Results shown in (G) represent data from three experiments. Results are presented as the mean ± SD and significance is defined as * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Binding Assay, Mutagenesis, Incubation, Control, Infection, Sonication, Immunoprecipitation, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: HspBP1 inhibits NF-κB mediated activation of HIV-1 transcription by obstructing p65-recruitment on LTR promoter. ( A ) HspBP1 competes with p65 for regulation of LTR driven gene-expression. HEK293T cells were transfected with indicated plasmids. Luciferase assays were performed 36 h post-transfection. ( B ) HspBP1 inhibits p50/p65-mediated activation of LTR promoter in a dose-dependent manner. HEK293T cells were co-transfected with the indicated plasmids. HspBP1-construct was transfected in three doses (0.25–0.75 ug). Luciferase assays were performed 36 h post-transfection. Expression of various constructs used in the assay was confirmed by immunoblotting. ( C ) HspBP1 over-expression diminishes the occupancy of p65 at LTR promoter. HEK293T cells were co-transfected with either control DNA and pNL4-3 or HspBP1 and pNL4-3. 48 h post-transfection, cells were fixed and chromatin was prepared followed by immunoprecipitation with p65 antibody or IgG control antibody. qRT-PCR analysis was performed with indicated primers spanning three different regions of LTR promoter. ( D ) HspBP1 knockdown causes an increase in p65-mediated induction of LTR promoter. HEK293T cells were transfected with either control siRNA or HspBP1 siRNA. 24 h post transfection, LTR promoter, Tat and p65 were over-expressed as indicated and luciferase assay was performed after 24 h of second transfection. Results represent data from three experiments and presented as the mean ± SD (* P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Activation Assay, Gene Expression, Transfection, Luciferase, Construct, Expressing, Western Blot, Over Expression, Control, Immunoprecipitation, Quantitative RT-PCR, Knockdown

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: HspBP1 inhibits activation of latently infected cells. Activation of latently infected cells [( A ) ACH2 and ( B ) U1] leads to depletion in HspBP1 levels. ACH2 and U1 were stimulated with PMA (50ng/ml) and TNFα (10 ng/ml). 36 h post-treatment, cells were harvested for RNA and protein lysate preparation. Expression of HspBP1 was analyzed by qRT-PCR and immunoblotting. ( C ) HspBP1 over-expression inhibits virus production whereas knockdown causes an increase in virus production in ACH2 cells. Nucleofection was performed to over-express or knockdown HspBP1 in these cell lines. 24 h post-nucleofection, cells were stimulated with PMA (50 ng/ml) or TNFα (10 ng/ml). 24 h post-activation, p24 ELISA was performed with the supernatant to determine the amount of virus produced. HspBP1 over-expression and knockdown was confirmed by immunoblotting. ( D ) HspBP1 over-expression inhibits virus production whereas knockdown causes an increase in virus production in U1 cells. The results shown in (A), (B) and (C) are representative of three experiments. (D) represents data from two experiments. Error bars represent the mean ± SD values and significance is defined as ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Activation Assay, Infection, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Virus, Knockdown, Enzyme-linked Immunosorbent Assay, Produced

Journal: Nucleic Acids Research

Article Title: HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

doi: 10.1093/nar/gkv1151

Figure Lengend Snippet: Proposed mechanistic model showing restriction of HIV-1 by HspBP1. Left, HIV-1 infection causes down-modulation of HspBP1 expression. Therefore, HspBP1 cannot inhibit HIV-1 production, consequently leading to efficient virus production. Right, when HspBP1 is over-expressed in an infected cell, HspBP1 interacts with NF-κB enhancer region (κB sites) on the HIV-1 LTR promoter and inhibits LTR driven gene-expression. The binding of HspBP1 to κB sites obliterates the binding of NF-κB hetero-dimer (p50/p65) to the same region, leading to repression in HIV-1 transcription.

Article Snippet: HspBP1 antibody (NBP201061) used in EMSA was obtained from Novus biological, USA.

Techniques: Infection, Expressing, Virus, Gene Expression, Binding Assay

Journal: BioMed Research International

Article Title: Improvement of Inflammation through Antioxidant Pathway of Gardeniae Fructus 50% EtOH Extract (GE) from Acute Reflux Esophagitis Rats

doi: 10.1155/2020/4826176

Figure Lengend Snippet: Western blot analysis of (a) survivin, (b) cytochrome c, (c) caspase-3, (d) Bax, and (e) Bcl-2 expressions. Nor: normal rats, Con: reflux esophagitis control rats, GL: GE 50 mg/kg treated reflux esophagitis rats, and GH: GE 100 mg/kg treated reflux esophagitis rats. (f) All data are expressed means ± SEM, ( n = 6) rats per group. Significance: ∗ p < 0.05, ∗∗ p < 0.01 versus RE control rat values.

Article Snippet: Mouse monoclonal antibodies against survivin (1 : 1,000, NB 500-205) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Reflux, Control