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