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: The Journal of Biological Chemistry

Article Title: Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones

doi: 10.1074/jbc.RA119.012449

Figure Lengend Snippet: Hsp110 NEFs show increased interaction with HSPA1A. A–C, interaction of GFP-HSPA1A or GFP-HSPA1L with NEFs of the BAG (A), HSPBP1 (B), or Hsp110 (C) families, in the presence of SOD1A4V. GFP-HSPA1A or GFP-HSPA1L were transiently expressed in HEK293 cells for 48 h with either HA-BAG1 or FLAG-BAG3 (A), untagged-HSPBP1 (B), and V5-tagged HSPH1, HSPH2, or HSPH3 (C). GFP Nanotrap was used for native immunoprecipitation (IP) of the Hsp70s. Western blots using the indicated antibodies are shown. The quantification graph of binding represents ratios of IP intensities of HA/GFP for BAG1 (n = 3) and FLAG/GFP for BAG3 (n = 2) (A), HSPBP1/GFP for HSPBP1 (n = 3) (B), and V5/GFP for HSPH1–3 (n = 3 for each) (C), all relative to HSPA1A measurements. In all graphs, error bars with S.E. *, p = 0.01–0.05; **, p = 0.001–0.01; ***, p < 0.001.

Article Snippet: Proteins were transferred onto polyvinylidene difluoride membranes and blotted with the primary antibodies: GFP (JL-8, 632381, Clontech); V5 (46-0705, Invitrogen); SOD1 (FL-154, sc-11407, Santa Cruz); FLAG (M2, 035K6196, Sigma); HSPH1/HSP105 (EPR4576, ab109624, Abcam); HSPH2/HSPA4 ( EPR14166 , ab185962, Abcam); HSPH3/HSPA4L (ab87241, Abcam); α-tubulin (B-5-1-2, T5168, Sigma); β-actin (8H10D10, Cell Signaling); HSPBP1 (1D5, NBP2–01168, Novus Biologicals); Hsp90 (total, 4F3-E8 SMC-149, StressMarq; Hip ([11A6], sc-136175, Santa Cruz); HOP (STI1, D-6; sc-390203, Santa Cruz); CHIP ([C10], sc-133083, Santa Cruz), and HA-peroxidase (12013819001, Roche).

Techniques: Immunoprecipitation, Western Blot, Binding Assay

Journal: PeerJ

Article Title: Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

doi: 10.7717/peerj.1530

Figure Lengend Snippet: (A) HspBP1 and hsc70 organization. Domains are depicted for HspBP1 and hsc70, numbers denote amino acid residues. The regions involved in the co-chaperone/chaperone interactions are marked; they are based on the crystal structure . NLS denotes the nucleolar localization sequence . Hsc70 and HspBP1can be modified posttranslationally; some of these modifications occur in the chaperone/co-chaperone interaction sites ( , ). (B) HeLa cells were grown at 37 °C or exposed to heat shock, followed by stress recovery. Representative confocal images for the immunolocalizations of HspBP1 and hsc70 are shown. Nuclei were detected with DAPI; an overlay of DAPI and HspBP1 images demarcates the nuclear compartments. Size bars are 20 µm. (C) Pixel intensities were quantified in the nucleus or cytoplasm, and the nucleocytoplasmic ratio was calculated. Results are shown as means + SEM for three independent experiments. Significant differences were identified by One-Way ANOVA, using unstressed control cells as the reference; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Samples were incubated for 1 h with primary antibodies against hsc70 (diluted 1:1,000; Stressgen SPA-815), HspBP1 (diluted 1:200; Santa Cruz sc-34252) or Crm1 (diluted 1:200, sc-5595).

Techniques: Sequencing, Modification, Control

Journal: PeerJ

Article Title: Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

doi: 10.7717/peerj.1530

Figure Lengend Snippet: Posttranslational modifications in hsc70/HspBP1 interaction sites. PhosphoSitePlus ( Hornbeck et al., 2015 ) reports more than 130 modifications for human hsc70 and 12 for human HspBP1. Table 1 lists only modifications that are located in hsc70/HspBP1 binding sites; they include phosphorylation (p) and ubiquitination (ub). Note that PhosphoSitePlus numbering is for the HspBP1 isoform that contains 362, rather than 359 amino acid residues as depicted in Fig. 1 and used to solve the crystal structure ( Shomura et al., 2005 ). The 362 amino acid isoform contains three additional glycine residues inserted after residue 30 of the 359 amino acid isoform.

Article Snippet: Samples were incubated for 1 h with primary antibodies against hsc70 (diluted 1:1,000; Stressgen SPA-815), HspBP1 (diluted 1:200; Santa Cruz sc-34252) or Crm1 (diluted 1:200, sc-5595).

Techniques: Binding Assay, Phospho-proteomics, Ubiquitin Proteomics, Residue

Journal: PeerJ

Article Title: Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

doi: 10.7717/peerj.1530

Figure Lengend Snippet: HeLa cells were grown under non-stress control conditions (Ctl) or exposed to heat shock (HS), followed by 3 h recovery (3 h rec). Crude extracts were examined by Western blotting with antibodies against HspBP1, hsc70 or actin. Enhanced chemiluminescence signals were quantified and normalized to actin. The graph shows means + SEM for three independent experiments. There were no significant changes for the abundance of HspBP1 or hsc70.

Article Snippet: Samples were incubated for 1 h with primary antibodies against hsc70 (diluted 1:1,000; Stressgen SPA-815), HspBP1 (diluted 1:200; Santa Cruz sc-34252) or Crm1 (diluted 1:200, sc-5595).

Techniques: Control, Western Blot

Journal: PeerJ

Article Title: Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

doi: 10.7717/peerj.1530

Figure Lengend Snippet: Quantitative image analysis was performed for control, heat-shocked and recovering cultures. Hsc70/HspBP1 data sets were obtained for the nucleus and cytoplasm of individual cells. Graphs depict single-cell results for three independent experiments. At least 115 cells were examined for each condition. Linear regression trendlines are included in the plots; functions and R 2 values for the best fit are shown at the right margin.

Article Snippet: Samples were incubated for 1 h with primary antibodies against hsc70 (diluted 1:1,000; Stressgen SPA-815), HspBP1 (diluted 1:200; Santa Cruz sc-34252) or Crm1 (diluted 1:200, sc-5595).

Techniques: Control

Journal: PeerJ

Article Title: Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

doi: 10.7717/peerj.1530

Figure Lengend Snippet: (A) The table depicts r values calculated for the pixel intensities of the hsc70/HspBP1 pair. Results are shown for the ratio nucleus/cytoplasm (N/C), the nucleus (Nuc) and cytoplasm (Cyt). (B) Graphical representation of r values for different experimental conditions.

Article Snippet: Samples were incubated for 1 h with primary antibodies against hsc70 (diluted 1:1,000; Stressgen SPA-815), HspBP1 (diluted 1:200; Santa Cruz sc-34252) or Crm1 (diluted 1:200, sc-5595).

Techniques:

Journal: PeerJ

Article Title: Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

doi: 10.7717/peerj.1530

Figure Lengend Snippet: (A) Areas of overlapping distribution were identified for Hsc70/HspBP1 and HspBP1/Hsc70 as described in Figure S2. A minimum of ten different cells was analyzed for each data point. Significant differences were identified by One-way ANOVA combined with Bonferroni post-hoc analysis. Pairwise comparisons were performed with control samples as reference; ∗ , p < 0.05; ∗∗ , p < 0.01. (B) Changes in the HspBP1/Hsc70 colocalization are shown both for the cytoplasm (outer ring of the pie graph) and nucleus (inner ring) for all conditions. In pixels containing HspBP1, hsc70 was present (colocalization, purple) or absent (no colocalization, grey). Numbers denote the percentage of HspBP1/Hsc70 colocalization.

Article Snippet: Samples were incubated for 1 h with primary antibodies against hsc70 (diluted 1:1,000; Stressgen SPA-815), HspBP1 (diluted 1:200; Santa Cruz sc-34252) or Crm1 (diluted 1:200, sc-5595).

Techniques: Control

Journal: Cell & Bioscience

Article Title: Heat shock protein 70 protects mouse against post-infection irritable bowel syndrome via up-regulating intestinal γδ T cell’s Th17 response

doi: 10.1186/s13578-018-0237-z

Figure Lengend Snippet: Protein and mRNA expression of HSP 70. a , b Expression of HSP 70 detected by Western blot; c , d Expression of HSP 70 detected by RT-PCR. *p < 0.05 compared with control group; △, p < 0.05 compared with both HSP70 group and IP-IBS group

Article Snippet: HSP70 protein level was measured by Western blot (Wuhan Boster Coporation, Wuhan, China).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Cell & Bioscience

Article Title: Heat shock protein 70 protects mouse against post-infection irritable bowel syndrome via up-regulating intestinal γδ T cell’s Th17 response

doi: 10.1186/s13578-018-0237-z

Figure Lengend Snippet: γδ T cells’ Th17 response and IL-17 production by γδ T cells. a Purified γδ T cells were stained with Anti-TCRγ/δ-FITC antibodies and runned on FACS; b γδ T cells’ proliferation index was measured; c Annexin V-FITC/PI apoptosis Kit was used to determine the apoptosis rate; d The surface molecular expression of CD62L and CD69 was detected by FACS; e The production of IL17 by γδ T cells stimulated by HSP70 was measured by ELISA

Article Snippet: HSP70 protein level was measured by Western blot (Wuhan Boster Coporation, Wuhan, China).

Techniques: Purification, Staining, Expressing, Enzyme-linked Immunosorbent Assay