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
Proteintech is a verified supplier 
