Journal: Scientific Reports

Article Title: Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation

doi: 10.1038/srep08805

Figure Lengend Snippet: (A), CHO cells were treated with A23187 or tunicamycin for 12 h. Cells were then harvested for immunoblotting with anti-22C11 (APP), anti-USP25, anti-HRD1, anti-Bip, and anti-β-actin antibodies. (B), CHO cells pre-incubated with MG132 or vehicle for 30 min were exposed to A23187 for 12 h. Whole-cell extract of CHO cells was subjected to immunoprecipitation (IP) with anti-USP25 antibody and then immunoblotted with anti-22C11 (APP), anti-USP25, anti-HRD1, and anti-β-actin antibodies. (C), CHO cells were treated with A23187 for 12 h after transfection with USP25. The total cell lysates were analyzed by western blotting with the antibodies. For each experiment, APP level was quantified by densitometry and normalized to β-actin loading control. Statistical significance was analyzed by one-way ANOVA followed by a Tukey's multiple-comparison test. (n = 3. *P < 0.05 versus vehicle). All the gels were run under the same experimental conditions. Full-length images are presented in the .

Article Snippet: The following antibodies were used for immunodetection: anti-22C11, Nicastrin (Millipore, Billerica, MA, USA), 6E10 (Signet, Dedham, MA, USA), sAPPβ (Covance, Princeton, NJ, USA), anti-Flag (Sigma), HA (Cell Signaling Technology, Seoul, Korea), USP25 (Santa Cruz, Dallas, TX, USA), HRD1 (Abgent, Seoul, Korea), Calnexin (Enzo, Farmingdale, NY, USA), LC3B (Novus, Littleton, CO, USA), GAPDH (Abcam, Seoul, Korea), β-actin and tubulin (Sigma-Aldrich).

Techniques: Western Blot, Incubation, Immunoprecipitation, Transfection

Journal: Frontiers in Veterinary Science

Article Title: The H protein of attenuated canine distemper virus is degraded via endoplasmic reticulum-associated protein degradation

doi: 10.3389/fvets.2023.1214318

Figure Lengend Snippet: The degradation of CDV 851H protein via ERAD. (A) The inhibition of ERAD by Eevarestatin I increased the CDV 851H protein level. 293T cells were transfected with Flag-CDV H for 24 h, then were treated with Eevarestatin I (4 μM) for 4 h. The cell lysates were analyzed by Western blot using anti-Flag and anti-GAPDH antibodies. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. (B) Knockdown of Hrd1 increased the CDV 851H protein level. After transfecting 293T cells with siRNA for Hrd1 or si-NC for 24 h, the cells were transfected with H protein of CDV 851 and CDV NJ(11)2 for another 24 h. Western blot was used to detect the CDV H protein level and confirm the silencing efficacy of Hrd1-targeted siRNA. The gray values of protein bands were analyzed by ImageJ software and the ratio of target protein gray values to GAPDH was calculated. All results are presented as means ± SD obtained from at least three independent sample preparations. * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies were obtained commercially: anti-Flag mouse monoclonal antibody (F1804, Sigma), anti-ATF6 rabbit polyclonal antibody (DF6009, Affinity), anti-Hrd1 rabbit polyclonal antibody (A2605, Abclonal), anti-HA mouse monoclonal antibody (BD-PM2095, Biodragon), anti-GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech), HRP-conjugated goat anti-mouse IgG (BF03001, Biodragon), and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (A0568, Beyotime).

Techniques: Inhibition, Transfection, Western Blot, Software, Knockdown