Journal: bioRxiv

Article Title: Disulfide Crosslinking Induces Rapid Degradation of Arc/Arg3.1 via Hsp70-Mediated Ubiquitin Ligase Pathway

doi: 10.1101/2025.07.20.665809

Figure Lengend Snippet: (A) HSF1-/- cells exhibit reduced expression of heat shock inducible Hsps. WT and HSF1-/- MEF cells were exposed to heat shock at 45 °C for 15 min and allowed to recover at 37 °C for the indicated times. Hsp90, Grp78, Hsc/Hsp70, Hsp60, Hsp40, and Hsp27 were detected by Western blot analysis with their specific antibodies. (B, C) Hsp70 participates in UPS-dependent Arc/Arg3.1 degradation. HSF1-/- MEF cells overexpressing Flag-Hsp70 were exposed to heat shock at 45 °C for 15 min and allowed to recover at 37 °C for the indicated times. Flag-Hsp70 and Arc/Arg3.1 were detected by Western blot analysis using their specific antibodies. Representative Western results were selected from triplicated experiments. Quantified results of triplicated Western images are shown in (C). (D) Hsp70 interacts with Arc/Arg3.1. HEK293T cells were transfected with GFP-Hsp70 and Flag empty vector or Flag-Arc, and the cell lysates were immunoprecipitated using an anti-Flag antibody. The immune complex was analyzed by Western analysis using anti-GFP and anti-Flag antibodies. (E) Arc/Arg3.1 primarly binds to Hsc/Hsp70. Following transfection of HEK293T cells with either Flag empty vector or Flag-Arc, the whole cell lysates were immunoprecipitated using an anti-Flag antibody. Western analysis using anti-Hsc/Hsp70, anti-Hsp40, and anti-Hsp27 antibodies was used to examine the immunological complex. * was an antibody’s light chain. (F, G) CHIP induces Arc/Arg3.1 degradation by interacting with Hsp70. HEK293T cells overexpressing Flag-Arc, GFP-Hsp70, and His-ubiquitin were co-transfected with HA-CHIP WT or mutants. Cells were analyzed by Western blot with anti-Flag, anti-HA, and anti-GFP antibodies. Representative Western results were selected from triplicated experiments. Quantified results of triplicated Western images are shown in (G). Data are presented as the mean ± S.D. of triplicated experiments (t-test; # < 0.01, **P < 0.001, *** < 0.0001). (H) Arc/Arg3.1 proteins are stabilized by CHIP knockdown. After being depleted of either control or CHIP, MEF cells were exposed to heat shock at 45°C for 15 min and then allowed to recover at 37°C for 9 h.

Article Snippet: Mouse monoclonal Arc antibody (E-7) (sc-55475), mouse monoclonal Arc antibody (C-7) (sc-17839), mouse monoclonal anti-β-Actin (C4) (sc-47778), mouse monoclonal HSP27 antibody (sc-13132), and mouse monoclonal α-tubulin antibody (sc-8035) were purchased from Santa Cruz: Rabbit polyclonal GAPDH antibody (LF-PA0018) and mouse monoclonal HA antibody (LF7H5) (LF-MA0048) from Ab Frontier: Mouse monoclonal Hsc/Hsp70 antibody (ADI-SPA-820) and mouse monoclonal HSF1 antibody (ADI-SPA-901) from Enzo: Mouse monoclonal FLAG antibody (F3165) from Sigma Aldrich: Mouse monoclonal GFP antibody (A-11120) from Invitrogen: Streptavidin-HRP (3999), rabbit monoclonal BiP antibody (C50B12) (3177), and rabbit polyclonal Hsp40 antibody (4868) from Cell signaling: Mouse monoclonal Hsp90 antibody (ab13492), rabbit polyclonal Hsp60 antibody (ab13492), and rabbit polyclonal Hsp60 antibody (ab46798) from Abcam: Goat mouse IgG-HRP conjugate antibody (170–5047) and goat rabbit IgG-HRP conjugate antibody (170–6515) from Bio-Rad.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Knockdown, Control

Journal: Poultry science

Article Title: HSP60 inhibits DF-1 apoptosis through its mitochondrial signal peptide.

doi: 10.1016/j.psj.2024.104571

Figure Lengend Snippet: Figure 1: DF-1-HSP60-KO in the DF-1 cell line. (A) Plasmid mapping of Px459M (pSpCas9- 2A-Puro-MCS) and EZ-GuideXH. (B) Results of PCR analysis of recombinant plasmid and double enzyme digestion. (Left) PCR analysis of recombinant plasmids. (Right) Recombinant plasmids were verified by double digestion with Xho I and Hind III enzymes. (C) Comparative results of HSP60 partial gene in monoclonal DF-1-HSP60-KO cell line and DF-1 cells. (D) Results of Western Blot assay of DF-1-HSP60-KO cells. After the cells were cultured in flat dishes for 24 h, relevant cellular protein samples were collected and then analyzed by Western Blot using HSP60 protein antibody.

Article Snippet: All the antibodies were procured from Proteintech and included HSP60 polyclonal (15282-1-AP, 1:10000), β-actin (81115-1- RR, 1:20000), BAX polyclonal (50599-2-lg, 1:10000), BAK polyclonal (29552-1-AP, 1:12000), Bcl-2 polyclonal (12789-1-AP, 1:10000), and Caspase 3 polyclonal (19677-1- AP, 1:2000) antibodies.

Techniques: Plasmid Preparation, Recombinant, Western Blot, Cell Culture

Journal: Poultry science

Article Title: HSP60 inhibits DF-1 apoptosis through its mitochondrial signal peptide.

doi: 10.1016/j.psj.2024.104571

Figure Lengend Snippet: Figure 2: The influence of HSP60 knockout on cell apoptosis. (A) ELISA was performed to determine the effect of HSP60 knockout on apoptosis. Levels of apoptosis induced by Bardoxolone MethyI at 4 h (left) and 6 h (right). (B) Detection of apoptosis level after knockout of HSP60 by flow cytometry. (C) Statistical results of apoptosis levels determined by flow cytometry. *p < 0.05; **p < 0.01, ***p < 0.001, ns p > 0.05.

Article Snippet: All the antibodies were procured from Proteintech and included HSP60 polyclonal (15282-1-AP, 1:10000), β-actin (81115-1- RR, 1:20000), BAX polyclonal (50599-2-lg, 1:10000), BAK polyclonal (29552-1-AP, 1:12000), Bcl-2 polyclonal (12789-1-AP, 1:10000), and Caspase 3 polyclonal (19677-1- AP, 1:2000) antibodies.

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Poultry science

Article Title: HSP60 inhibits DF-1 apoptosis through its mitochondrial signal peptide.

doi: 10.1016/j.psj.2024.104571

Figure Lengend Snippet: Figure 3: Effect of DF-1-HSP60-KO on apoptosis signaling pathway. (A) Validation of Bax, P53, Bcl-2, Caspase3, and Bak transcript levels in DF-1-HSP60-KO cells. (B) Verification of Bcl-2, HSP60, Bax, Caspase3, and Bak protein expression in DF-1-HSP60-KO cells. (C) Statistical results of relative β-actin expression levels of Bcl-2, Bax, HSP60, Bak, and Caspase3 proteins in DF-1-HSP60-KO cells. *p < 0.05; **p < 0.01, ***p < 0.001, ns p > 0.05.

Article Snippet: All the antibodies were procured from Proteintech and included HSP60 polyclonal (15282-1-AP, 1:10000), β-actin (81115-1- RR, 1:20000), BAX polyclonal (50599-2-lg, 1:10000), BAK polyclonal (29552-1-AP, 1:12000), Bcl-2 polyclonal (12789-1-AP, 1:10000), and Caspase 3 polyclonal (19677-1- AP, 1:2000) antibodies.

Techniques: Biomarker Discovery, Expressing

Journal: Poultry science

Article Title: HSP60 inhibits DF-1 apoptosis through its mitochondrial signal peptide.

doi: 10.1016/j.psj.2024.104571

Figure Lengend Snippet: Figure 4: The effect of HSP60 MIT on cell apoptosis. (A) Structure prediction of HSP60 MIT mutant protein. (B) Recombinant plasmid sequencing results (top). DF-1-HSP60-KO cells were transfected with empty vector, wild-type pEGFP-HSP60 (WT), and mutant pEGFP-TB plasmid. Fluorescence microscopic analysis of transfected cells after 24 hours (down). (C) ELISA was performed to evaluate the effect of HSP60 MIT mutation on apoptosis. Apoptosis levels induced by Bardoxolone MethyI at (left) 4 h and (right) 6 h. (D) Apoptosis rates in HSP60 MIT mutants were detected by flow cytometry in DF-1-HSP60-KO cells. (E) Statistical results of apoptosis levels were assessed by flow cytometry. Compared with the pEGFP-C1 group samples in the same group, *p < 0.05; **p < 0.01, ***p < 0.001, ns p > 0.05. Compared with the pEGFP-HSP60 and pEGFP-TB group samples in the same group, # p < 0.05, ## p < 0.01, ### p < 0.001, ns p > 0.05.

Article Snippet: All the antibodies were procured from Proteintech and included HSP60 polyclonal (15282-1-AP, 1:10000), β-actin (81115-1- RR, 1:20000), BAX polyclonal (50599-2-lg, 1:10000), BAK polyclonal (29552-1-AP, 1:12000), Bcl-2 polyclonal (12789-1-AP, 1:10000), and Caspase 3 polyclonal (19677-1- AP, 1:2000) antibodies.

Techniques: Mutagenesis, Recombinant, Plasmid Preparation, Sequencing, Transfection, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Poultry science

Article Title: HSP60 inhibits DF-1 apoptosis through its mitochondrial signal peptide.

doi: 10.1016/j.psj.2024.104571

Figure Lengend Snippet: Figure 5: The effect of HSP60 MIT on apoptosis signaling pathway. (A) Validation of Bax, HSP60, Bak, Bcl-2, Caspase3, P53 transcript levels at different times after HSP60 MIT mutation

Article Snippet: All the antibodies were procured from Proteintech and included HSP60 polyclonal (15282-1-AP, 1:10000), β-actin (81115-1- RR, 1:20000), BAX polyclonal (50599-2-lg, 1:10000), BAK polyclonal (29552-1-AP, 1:12000), Bcl-2 polyclonal (12789-1-AP, 1:10000), and Caspase 3 polyclonal (19677-1- AP, 1:2000) antibodies.

Techniques: Biomarker Discovery, Mutagenesis