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: Redox Biology

Article Title: LACTB suppresses liver cancer progression through regulation of ferroptosis

doi: 10.1016/j.redox.2024.103270

Figure Lengend Snippet: HSPA8 is a target of LACTB. A. qRT-PCR testing the effects of LACTB overexpression and knockout on HSPA8 mRNA levels. B. Western blot testing the indicated protein levels after LACTB overexpression in SK-HEP-1 cells. C. Western blot testing the indicated protein levels in LACTB −/− cells reexpressing LACTB. D. Western blot testing the indicated protein levels in LACTB-expressing cells transfected with HSPA8-expressing plasmid. E. Western blot testing the indicated protein levels in LACTB −/− cells transfected with si-HSPA8. F–I. Detection of effects of LACTB overexpression or in combination with HSPA8 overexpression on Fe 2+ , lipid peroxidation, MDA levels and GSH/GSSG ratio in cells. Scale bar = 25 μm. J,K. Detection of effects of LACTB knockout or in combination with HSPA8 silencing on Fe 2+ and lipid peroxidation levels in HepG2 and SK-HEP-1 cells. Scale bar = 25 μm. L. Kaplan-Meier plotter database showing the overall survival rate of liver cancer patients with high or low HSPA8 expression. M. Xenograft tumour model testing the in vivo effects of LACTB or LACTB + HSPA8 on SK-HEP-1 cell growth. Scale bar = 1 cm. Student's t -test was used for A (left panel), one-way ANOVA with Tukey post-hoc test was used for A (right panel), F–K, M (right panel), and two-way ANOVA with Tukey post-hoc test was used for M (middle panel).

Article Snippet: The sections were then incubated with anti -LACTB (#18195-1-AP, Proteintech), anti -p53 (wild-type) (#MABE339, Sigma-Aldrich), and anti -HSPA8 (#M01024-1, BOSTER) primary antibodies and a DAKO peroxidase-labelled secondary antibody.

Techniques: Quantitative RT-PCR, Over Expression, Knock-Out, Western Blot, Expressing, Transfection, Plasmid Preparation, In Vivo

Journal: Redox Biology

Article Title: LACTB suppresses liver cancer progression through regulation of ferroptosis

doi: 10.1016/j.redox.2024.103270

Figure Lengend Snippet: LACTB drives ferroptosis via regulating the p53/HSPA8 axis. A. qRT-PCR testing HSPA8 mRNA levels in LACTB-expressing Huh7 and Hep3B cells. B, C. qRT-PCR and Western blot testing HSPA8 mRNA and protein levels in LACTB-expressing cells transfected with si-p53. D. Western blot testing p53 protein expression in LACTB-expressing SK-HEP-1 cells treated with 100 μg/mL cycloheximide for the indicated time. E. Western blot testing p53 ubiquitination levels in LACTB-expressing SK-HEP-1 cells. F. Western blot testing HSPA8 protein expression in LACTB-expressing p53 +/+ Hep3B cells. G. Luciferase reporter assay testing HSPA8 promoter activity in LACTB-expressing cells transfected with si-p53. H. p53 binding sites on HSPA8 promoter among different species. I. The schema showing the wild-type or mutant p53 binding motifs on HSPA8 or p21 promoter, and primer design positions for ChIP assay. J. ChIP assay testing the binding of p53 on the indicated regions of HSPA8 promoter. K. DNA pull-down assay using wild-type or mutant HSPA8 promoter probe, followed by Western blot analysis of p53 protein expression. L. Generation of SK-HEP-1 cells with endogenous mutation of p53 binding site in HSPA8 promoter using CRISPR/Cas9 gene editing technology with the indicated ssODN and gRNA. M. CCK-8 testing the viability of wild-type or mutant SK-HEP-1 cells treated with erastin and RSL3. N, O. Liperfluo and FerroOrange staining testing lipid peroxidation and Fe 2+ levels in wild-type or mutant SK-HEP-1 cells. Scale bar = 25 μm. P. Western blot testing HSPA8 protein levels in wild-type or mutant SK-HEP-1 cells with LACTB or p53 overexpression. Q. IHC staining of LACTB, wild-type (wt) p53 and HSPA8 in liver cancer tissue microarray, followed by analysis of their expression correlations. Scale bar = 50 μm. One-way ANOVA with Tukey post-hoc test was used for B and G. Student's t -test was used for J, N, O.

Article Snippet: The sections were then incubated with anti -LACTB (#18195-1-AP, Proteintech), anti -p53 (wild-type) (#MABE339, Sigma-Aldrich), and anti -HSPA8 (#M01024-1, BOSTER) primary antibodies and a DAKO peroxidase-labelled secondary antibody.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Ubiquitin Proteomics, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Mutagenesis, Pull Down Assay, CRISPR, CCK-8 Assay, Staining, Over Expression, Immunohistochemistry, Microarray

Journal: Redox Biology

Article Title: LACTB suppresses liver cancer progression through regulation of ferroptosis

doi: 10.1016/j.redox.2024.103270

Figure Lengend Snippet: LACTB potentiates the response of liver cancer to lenvatinib. A, B. FerroOrange and liperfluo staining testing Fe 2+ and lipid peroxidation levels in HepG2 and SK-HEP-1 cells treated with lenvatinib, respectively. Scale bar = 25 μm. C. Western blot testing LACTB protein expression in HepG2 and SK-HEP-1 cells treated with lenvatinib. D-G. FerroOrange, liperfluo staining, CCK-8 and colony formation assays respectively testing Fe 2+ , lipid peroxidation levels, viability and cloning ability in lenvatinib-treated cells with LACTB overexpression or knockout. H, I. Xenograft tumour model testing the in vivo effects of LACTB overexpression or knockout on the anti-tumour effect of lenvatinib. Scale bar = 1 cm. J. Western blot testing the indicated protein expression in the indicated groups. K. The proposed model showing that LACTB induced by lenvatinib promotes ferroptosis by increasing p53 protein stability and inhibiting HSPA8-mediated anti -ferroptosis pathway. One-way ANOVA with Tukey post-hoc test was used for A, B, D-G, I.

Article Snippet: The sections were then incubated with anti -LACTB (#18195-1-AP, Proteintech), anti -p53 (wild-type) (#MABE339, Sigma-Aldrich), and anti -HSPA8 (#M01024-1, BOSTER) primary antibodies and a DAKO peroxidase-labelled secondary antibody.

Techniques: Staining, Western Blot, Expressing, CCK-8 Assay, Cloning, Over Expression, Knock-Out, In Vivo

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of ChAT with heat shock proteins HSC70, HSP70, and HSP90 is altered by mutation of N-terminal proline-rich motif in ChAT. (A) Immunoblots showing co-IP of ChAT with endogenous HSC70, HSP70 and HSP90 from HEK293 cells expressing either wild-type or P17A/P19A-ChAT. Control cells were transfected with empty vector. Using HEK293 cells, co-IP of P17A/P19A-ChAT with HSP70 (B) , HSP90 (C) and HSC70 (D) , respectively, is greater than that of wild-type ChAT ( *** p ≤ 0.001, Student's t -test, mean ± SEM, n = 4). (E) Co-IP of ChAT with endogenous HSC70 and HSP90 from mouse cholinergic SN56 cells expressing either wild-type or P17A/P19A-ChAT or CMS-related mutant proteins V18M- or A513T-ChAT. Control cells were transfected with empty vector. (F) Using SN56 cells, Co-IP of P17A/P19A-ChAT ( *** p ≤ 0.001) and V18M-ChAT ( * p ≤ 0.05), but not A531T-ChAT, with HSC70 is greater than that of wild-type ChAT (mean ± SEM, n = 5). (G) While there was a trend toward increased HSP90 interaction with P17A/P19A-ChAT ( p = 0.09), no significant differences were observed for HSP90 interaction with mutant ChAT compared to wild-type ChAT in SN56 cells (mean ± SEM, n = 5). Statistical analysis for (F) and (G) was performed by one-way ANOVA with Dunnett's post-hoc test. (H) Detection of in situ interactions of wild-type ChAT with endogenous HSC70 and HSP90 by proximity ligation assay (PLA) in SN56 cells. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with either mouse anti-HSC70 or mouse anti-HSP90 primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink in Situ Orange Kit (Sigma), in situ ChAT-HSP interactions were imaged by confocal microscopy. Positive in situ ChAT-HSP interactions where visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or had primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 3 independent experiments; scale bars are 10 μm.

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Expressing, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: ChAT interacts with the HSP-associated E3 ubiquitin ligase C-terminus of HSC70-interaction protein (CHIP). (A) Immunoblots showing co-IP of ChAT with FLAG-CHIP from SN56 cells co-expressing either wild-type or mutant ChAT protein with FLAG-tagged CHIP. Control cells were transfected with either empty vector or to express either wild-type ChAT or FLAG-CHIP alone. (B) Co-IP of ChAT with FLAG-CHIP is enhanced for P17A/P19A- ( *** p ≤ 0.001), V18M- ( *** p ≤ 0.001), and A513T-ChAT ( * p ≤ 0.05) as compared to wild-type ChAT (one-way ANOVA with Dunnett's post-hoc test, mean ± SEM, n = 5). (C) Co-IP of wild-type and mutant ChAT with endogenous CHIP following anti-ChAT co-IP from ChAT-expressing SN56 cells ( n = 3). (D) Detection of in situ ChAT interactions with endogenous CHIP by proximity ligation assay (PLA) in SN56 cells expressing wild-type ChAT. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with rabbit anti-CHIP primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink In Situ Orange Kit (Sigma), in situ ChAT-CHIP interactions were imaged by confocal microscopy. Positive in situ ChAT-CHIP interactions were visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 4 independent experiments; scale bars are 10 μm. (E) siRNA-mediated knock-down of CHIP has no effect on the steady-state protein levels of either wild-type or mutant ChAT. ChAT-expressing SN56 cells were co-transfected with 25 nM of either anti-CHIP siRNA or scramble-control siRNA for 72 h. Control cells were mock-transfected ( n = 4).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining