Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) In vivo evidence for Hsc70-Cx43 interaction. SDS-PAGE and Coomassie staining of proteins obtained in co-immunoprecipitation assay with anti-Cx43 antibodies using lysates prepared from HuH-7 cells. Arrows indicate the positions of Hsc70 and β-tubulin. Both proteins were identified by MALDI/Q-TOF mass spectrometry analysis. (b) Binding of Hsc70 to GST-Cx43CT. Proteins extracted from HuH-7 cells were mixed either with GST or with GST-Cx43CT residues 228-382 resin. After washing, the bound proteins were released and analyzed on 12% SDS-PAGE followed by western blotting (WB) with anti-Hsc70 antibody. Full-length blots are presented in . (c and d) Co-immunoprecipitation of Hsc70 with Cx43. Lysates were prepared from HuH-7 cells co-transfected with wild-type Cx43 and T7-tagged Hsc70 expression plasmids. Lysates were immunoprecipitated (IP) either by anti-Cx43 antibodies and control IgG (c) or by anti-T7 antibody and the IgG (d). The immunocomplexes were analyzed by western blotting (WB) using anti-T7 antibody (c) or anti-Cx43 antibodies (d). Full-length blots are presented in . (e) Co-localization of endogenous Hsc70 and Cx43. HuH-7 cells were fixed and stained with anti-Cx43 antibodies and anti-Hsc70 antibody. The nuclei were stained with TO-PRO3. Co-localization of both proteins was observed in the cytoplasm.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: In Vivo, SDS Page, Staining, Co-Immunoprecipitation Assay, Mass Spectrometry, Binding Assay, Western Blot, Immunoprecipitation, Transfection, Expressing

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) A schematic representation of GST-Cx43CT fusion proteins used as a ligand. Consensus protein domains and regions with putative signaling functions are indicated. Abbreviations in Cx43: TM1-4, transmembrane domains 1–4; JM, juxtamembrane domain that binds microtubules; Y265, Src phosphorylation site; PP, proline-rich sequence that binds the SH3 domain of v-Src; DLEI, carboxy-terminal sequence involved in ZO-1 binding. (b) Identification of Hsc70-binding domain in Cx43. Whole cell lysates of HuH-7 cells and various forms of the GST-Cx43CT fusion proteins were used for GST pulldown assays. Binding experiments and western blotting (WB) were carried out as described in . (c) A schematic representation of Hsc70 and its deletion mutants used for overexpression. (d) Identification of Cx43 binding domain in Hsc70. Expression vectors encoding various Hsc70s were transfected into HuH-7 cells, and binding experiments were carried out as described in . The immunocomplexes were analyzed by western blotting (WB) using anti-T7 antibody.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Sequencing, Binding Assay, Western Blot, Over Expression, Expressing, Transfection

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) Hsc70 interacts with cyclin D1 or Cx43. Co-immunoprecipitation of Hsc70 with cyclin D1 or Cx43. Lysates were prepared from HuH-7 cells co-transfected with T7-tagged Hsc70 and cMyc-tagged cyclin D1 or wild-type Cx43 expression plasmids. Lysates were immunoprecipitated (IP) either by anti-cMyc antibody and control IgG (left) or by anti-Cx43 antibodies and the IgG (right). The immunocomplexes were analyzed by western blotting (WB) using anti-T7 antibody. (b) Absence of interaction between cyclin D1 and Cx43. Co-immunoprecipitation of Cx43 with cyclin D1. Lysates were prepared from HuH-7 cells co-transfected with wild-type Cx43 and cMyc-tagged cyclin D1 expression plasmids. Lysates were immunoprecipitated (IP) either by anti-cMyc antibody and control IgG (left) or by anti-Cx43 antibodies and the IgG (right). The immunocomplexes were analyzed by western blotting (WB) using anti-Cx43 antibodies (left) or anti-cMyc antibody (right). (c) Competition assay of Cx43 and cyclin D1 for binding with Hsc70. HuH-7 cells were transfected with equivalent plasmids of both T7-tagged Hsc70 and cMyc-tagged cyclin D1, and with increasing amounts of Cx43. Lysates were immunoprecipitated (IP) by anti-T7 antibody. The immunocomplexes were analyzed by western blotting (WB) using anti-T7-tag, anti-Cx43, and anti-cMyc-tag antibodies.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Competitive Binding Assay, Binding Assay

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) BrdU incorporation assays were examined in HuH-7 cells. HuH-7 cells were transiently transfected with vectors encoding empty vector EGFP, Hsc70-EGFP, Cx43-EGFP alone, or both Hsc70- and Cx43-EGFP, respectively. Forty-eight hours after transfection, the cells were labeled with bromodeoxyuridine (BrdU) for 2 hr and subsequently fixed and stained with anti-BrdU antibody. Green indicates transfectants, and red indicates BrdU-incorporated cells. (b) The fraction of BrdU-positive cells over total number of transfected cells was determined. The data shown in (b) are means and SDs (error bars) of a representative experiment performed in triplicate at least. * p < 0.01. N.S.; not significant.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: BrdU Incorporation Assay, Transfection, Plasmid Preparation, Labeling, Staining

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) A schematic representation of Hsc70 and its splice variant, Hsc54, used for overexpression. (b) Pulldown assays with GST-Cx43CT fusion protein were performed using lysates prepared from Hsc70- or Hsc54-transfected HuH-7 cells. Expression vectors encoding Hsc70-EGFP and Hsc54-EGFP were transfected into HuH-7 cells, and binding experiments were carried out as described in . The bound proteins were analyzed by western blotting (WB) using anti-GFP antibody. (c) BrdU incorporation assays in HuH-7 cells. HuH-7 cells were transiently transfected with vectors encoding empty vector EGFP, Hsc54-EGFP, Cx43-EGFP alone, or both Hsc54- and Cx43-EGFP, respectively. BrdU incorporation and immunofluorescence staining were carried out as described in . (d) The fraction of BrdU-positive cells over total number of transfected cells. (e) Inhibition assay of protein-protein interaction. To inhibit the binding of proteins to Hsc70, HuH-7 cells were cultured with 10 μg/ml of 15-DSG for 24 hr. The fraction of BrdU-positive cells over total cells was determined. The data shown in (d) and (e) are means and SDs (error bars) of a representative experiment performed in triplicate at least. * p < 0.01. N.S.; not significant.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Variant Assay, Over Expression, Transfection, Expressing, Binding Assay, Western Blot, BrdU Incorporation Assay, Plasmid Preparation, Immunofluorescence, Staining, Inhibition, Cell Culture

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) Hsc70 translocated into the nucleus under heat shock. HuH-7 cells were heat shocked at 42°C for 2 hr and stained with anti-Hsc70 antibodies. (b) Overexpression of Cx43 prevented the nuclear translocation of Hsc70 under heat shock. HuH-7 cells were transfected with control vector EGFP or Cx43-EGFP. Forty-eight hours after transfection followed by heat shocked at 42°C for 2 hr, cells were stained with anti-Hsc70 antibodies. (c) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in Hsc70. After heat shock, at least 30 transfected HuH-7 cells of control EGFP or Cx43-EGFP were examined and quantified by confocal scanning microscopy. The data shown in (b) were plotted. The horizontal lines represent mean values. * p < 0.01.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Staining, Over Expression, Translocation Assay, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Connexin43 Functions as a Novel Interacting Partner of Heat Shock Cognate Protein 70

doi: 10.1038/srep02719

Figure Lengend Snippet: (a) Effect of Cx43 on nuclear accumulation of cyclin D1. HuH-7 cells were transfected with control plasmid mRFP or Cx43-mRFP, or both Cx43-mRFP and Hsc70-DsRed2. Immunofluorescence staining with anti-cyclin D1 antibodies was carried out. Nuclear accumulation of cyclin D1 was examined and quantified by confocal laser scanning microscopy. (b) Ratio of nuclear/cytoplasmic fluorescence intensities in cyclin D1. At least 30 transfected HuH-7 cells of control mRFP or Cx43-mRFP, or both Cx43-mRFP and Hsc70-DsRed2 were examined and quantified. The data shown in (a) were plotted. The horizontal lines represent mean values. * p < 0.01.

Article Snippet: The cells were then incubated with the following primary antibodies at room temperature for 2 hr: rabbit anti-Cx43 antibody (Sigma, 1:500); mouse anti-Hsc70 antibody (Santa Cruz, 1:200) or rabbit anti-Hsc70 antibody (StressMarq, Victoria, Canada, 1:250); rabbit anti-cyclin D1 antibody (Santa Cruz, 1:200); rabbit anti-p27 antibody (Santa Cruz, 1:200), and rabbit anti-Skp2 antibody (Santa Cruz, 1:200).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining, Confocal Laser Scanning Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Targeting hypoxia-induced circSTT3A decreases breast cancer stem cell formation via degradation of PGK1 protein and serine synthesis

doi: 10.1101/2023.04.28.538664

Figure Lengend Snippet: a. Silver staining of proteins pulled down by circSTT3a-specific biotin-labeled probe and control probe. HSP70 was ascertained as a candidate protein interacting with circSTT3A by RNA pull-down. b. The interaction between circSTT3A and HSP70 was verified by RIP and qRT-PCR in MDA-MB-231 and BT-549 cell lysate. c. FISH and IF co-staining indicated the co-localization of circSTT3A (red) and HSP70 (green) in MDA-MB-231 and BT-549 cells cultured in hypoxia (Scale bar, 25 μm). d-e . qRT-PCR and Western blotting were used to detect the effects of hypoxia-induced circSTT3A or ectopic circSTT3A overexpression on the expression of HSP70 in BC cells. f. Schematic diagram of HSP70 full-length protein and truncated protein. g. RNA pull-down experiment using biotin-labeled circSTT3A probe in hypoxic MDA-MB-231 cells expressing full-length or truncated HSP70, Western blot revealed the enrichmen of indicated full-length of HSP70 and its deletion mutants. h. RIP assays were performed with anti-Flag in hypoxic MDA-MB-231 cells transfected with indicated full-length or truncated HSP70 plasmids. i. Co-IP assays were carried out in hypoxic MDA-MB-231 cells under indicated conditions using anti-HSP70 and IgG control, followed by silver staining. j. The Co-IP experiment used anti-HSP70 or anti-PGK1 to analyze the direct interaction between HSP70 and PGK1 in MDA-MB-231, respectively. k. Co-IP using anti-Flag was applied to identify the interaction between the truncated HSP70 protein and PGK1 in MDA-MB-231 cells transfected with indicated full-length or truncated HSP70 plasmids. l. IF co-staining showed the co-localization of HSP70 (red) and PGK1 (green) in BC cells (Scale bar, 25 μm). Data were presented as the mean±SD, ***p<0.001.

Article Snippet: After washing with PBS, the cells were incubated with anti-HSP70 antibody at 4 °C overnight, then FITC-labeled goat anti-rabbit IgG was added (Bosterbio, China) at 37 °C for 2 h. DAPI was use to locate nucleus.

Techniques: Silver Staining, Labeling, Control, Quantitative RT-PCR, Staining, Cell Culture, Western Blot, Over Expression, Expressing, Transfection, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Targeting hypoxia-induced circSTT3A decreases breast cancer stem cell formation via degradation of PGK1 protein and serine synthesis

doi: 10.1101/2023.04.28.538664

Figure Lengend Snippet: a-b. mRNA and protein levels of PGK1 were determined by qRT-PCR (a) and Western blotting (b) in BC cells under indicated conditions. c. co-IP and WB were carried out in hypoxic MDA-MB-231 cells with or without circSTT3 and in normoxic MDA-MB-231 with or without ectopic circSTT3A expression using anti-HSP70 and anti-PGK1, IgG as negative controls. d. The expression of PGK1 was determined after transfection or co-transfection with the indicated vectors or siRNA in MDA-MB-231 cells by Western blotting. e. Western blot analysis of PGK1 expression in hypoxic and normoxic BC cells transfected with corresponding siRNA under MG-132 treatment. f. Ubiquitination of PGK1 was appraised in MG132-treated MDA-MB-231 cells under indicated conditions. g-h. The expression of PGK1 in BC tissues with low or high level of circSTT3A were evaluated by Western blotting (g) and qRT-PCR in 60 BC tissues (h) . i. Correlation analysis of circSTT3A and PGK1 in BC tissues. j-k. The representative images (j) and quantitative analysis (k) of mammosphere formation abilities were showed after transfection or co-transfection with the indicated vectors or siRNA. l. The representative images and volume analysis of xenograft tumors for each group (n=8). Data are presented as the mean±SD; *p<0.05, **p<0.01 and ***p < 0.001.

Article Snippet: After washing with PBS, the cells were incubated with anti-HSP70 antibody at 4 °C overnight, then FITC-labeled goat anti-rabbit IgG was added (Bosterbio, China) at 37 °C for 2 h. DAPI was use to locate nucleus.

Techniques: Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Expressing, Transfection, Cotransfection, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Targeting hypoxia-induced circSTT3A decreases breast cancer stem cell formation via degradation of PGK1 protein and serine synthesis

doi: 10.1101/2023.04.28.538664

Figure Lengend Snippet: a. Cellular 3-phosphoglycerate (3-PG), phosphoserine (pSer) and serine concentrations were measured by HPLC-MS in circSTT3A-knockdown, HSP70-knockdown and PGK1 knockdown MDA-MB-231 derived spheres and control spheres under hypoxia. b-c . Colony formation assays (b) , western blot analyses (c) of stemness-related proteins (CD44, c-Myc, and KLF4) in BC cells under indicated conditions (Scale bar, 200 µm). d-f . The expression of circSTT3A (d) , PGK1 (e) and serine (f) in the chemo-sensitive tissues (n=27) and chemo-resistant tissues (n=27) of BC patients. g-h . Representative pictures of xenograft tumors (n=8) (g) and xenograft tumor volume (h). i-j . Representative images showed PCNA IHC staining (i) , cell apoptotic status checked by TUNEL (i) , KLF4 and OCT4 levels (j) in each group of xenograft tumors (Scale bar, 100 µm). Data are presented as the mean±SD; *p<0.05, **p <0.01 and ***p < 0.001.

Article Snippet: After washing with PBS, the cells were incubated with anti-HSP70 antibody at 4 °C overnight, then FITC-labeled goat anti-rabbit IgG was added (Bosterbio, China) at 37 °C for 2 h. DAPI was use to locate nucleus.

Techniques: Knockdown, Derivative Assay, Control, Western Blot, Expressing, Immunohistochemistry, TUNEL Assay

Journal: Frontiers in Physiology

Article Title: Targeting Heat Shock Proteins Mitigates Ventilator Induced Diaphragm Muscle Dysfunction in an Age-Dependent Manner

doi: 10.3389/fphys.2016.00417

Figure Lengend Snippet: Western blot analyses of the Hsp72 protein expression normalized to actin contents in the diaphragm in control animals (individual rats: young n = 4 and old emphn = 2) compared with the age-matched animals exposed to CMV for 5 days with (individual rats: young n = 3 and old n = 2) and without BGP-15 (individual rats: young n = 3 and old n = 3) . (Black bars, young; white bars, old). Values are means + SEM. Significance level, * p < 0.05.

Article Snippet: Membranes were blocked with 3% non-fat milk powder in Tris-buffered saline containing Tween 20 for 1 h and incubated overnight at 4°C with Hsp72- (SMC-100B, StressMarq) and actin (sc-1616, Santa Cruz Biotechnology Inc.) primary antibodies and then with HRP secondary antibodies (anti-mouse, GE Healthcare, and anti-goat, Santa Cruz Biotech).

Techniques: Western Blot, Expressing

Journal: Microbiology Spectrum

Article Title: Chlamydia trachomatis Subverts Alpha-Actinins To Stabilize Its Inclusion

doi: 10.1128/spectrum.02614-22

Figure Lengend Snippet: α-Actinins are not required for actin scaffold formation. (A) Cells were transfected with nontargeting (Ctrl), human ACTN1 (α-actinin 1), or human ACTN4 (α-actinin 4) siRNA for 72 h and retransfected with equivalent amounts of siRNA for 72 h. Lysates were analyzed by Western blotting to assess the degree of α-actinin knockdown. HSP70 was used as a loading control. Knockdown efficiency was determined by measuring the ratio of α-actinin:HSP70 signal normalized to Ctrl siRNA, representative of 3 independent experiments. (B) Cells were transfected with Ctrl, α-actinin 4, or α-actinin 1 siRNA for 72 h and retransfected with equivalent amounts of siRNA for 24 h prior to infection with WT C. trachomatis L2 (MOI, 1) for 46 h. Cells were fixed and labeled with phalloidin (red), anti-MOMP (green), and Hoechst (DNA, gray). Asterisks denote inclusions. Scale bar, 25 μm. The white boxes represent a magnified section of the inclusion to show actin scaffolds (Zoom). (C) The graph represents the average percentage of inclusions with actin scaffolds from three independent experiments ± SD. A minimum of 100 inclusions were counted for each experiment. **, P < 0.01; ns, not significant.

Article Snippet: The following primary antibodies were used: anti-α-actinin 4 (mouse, sc-393495; Santa Cruz Biotechnology [SCBT]), anti-α-actinin 1 (rabbit, ab68194; Abcam), anti-α-actinin 4 (mouse, sc-390205; SCBT), anti-α-tubulin (mouse, T5168; Sigma), anti-α-tubulin (rat, sc-53029; SCBT), anti-acetylated α-tubulin (mouse, T6793; Sigma), anti-detyrosinated α-tubulin (rabbit, 48389; Abcam), ActiStain-488 (PHDG1; Cytoskeleton), ActiStain-555 (PHDH1-A; Cytoskeleton), anti-heat shock protein 70 (HSP70) (chicken, SPC-178D; StressMarq), anti-IncA (rabbit; T. Hackstadt), and anti-MOMP (goat, 1621; ViroStat).

Techniques: Transfection, Western Blot, Knockdown, Control, Infection, Labeling