Anti grp78

CEF cells were lysed in cell lysis buffer (Beyotime) and incubated on ice for 5 minutes. ALV-J gp85, REV env expression, Hsp70, GRP78 and CD81 were detected by simple western analysis with anti-NX0101 gp85, anti-SNV env antibody, anti-Hsp70 (Bioss), anti-GRP78 (Bioss) antibody and anti-CD81 (Bioss) antibody at a 1:200, 1:200, 1:1000, 1:1000 and 1:1000 dilution, respectively.

Tissue samples were fixed in 10% buffered formalin for 48 hours and then embedded in paraffin; 5‐μm slides were cut and dried overnight. Sections were de‐paraffinized and rehydrated prior to staining with hematoxylin and eosin and the indicated antibodies, including anti‐a‐SMA (Bioss, China, 1:100), anti‐Collagen I (Bioss, China, 1:100), anti‐ASIC1a (1:250, Alomone, USA), anti‐GRP78 (Bioss, China, 1:250,) and anti‐p‐AKT (Elabscience, China, 1:250). Samples were incubated with primary antibodies overnight at 4°C, secondary antibodies for 30 minutes. Slides were washed in PBS (Boster, Wuhan, China) 3 × 10 minutes after primary and secondary antibody incubations.

Formalin-fixed, paraffin-embedded SAT sections were used for immunohistochemical analysis and immunofluorescence investigations (n = 5 each), as previously described [23 (link)]. For the immunohistochemical analysis, we used anti-GRP78 (Abcam, Cambridge, MA, USA), Imgenex anti-ATF6 (Novus, Littleton, CO, USA), and anti-EPHX2 antibodies (OriGene Technologies, Inc., Rockville, MD, USA). The immunohistochemical analysis results were quantified using ImageScope software version 11.1 (Aperio, Vista, CA, USA), as previously reported [29 (link)]. For immunofluorescent staining, tissue sections were incubated with anti-GRP78 or anti-ATF6 antibody conjugated with Alexa Fluor-488 (Bioss, Woburn, MA, USA). EPHX2 tissue sections were incubated with anti-EPHX2 antibody (OriGene Technologies, Inc., Rockville, USA) and then incubated with Alexa Fluor-488–conjugated goat anti-mouse secondary antibody (Molecular Probes, ThermoFisher, USA). Nuclei were stained using 0.05% DAPI. The sections were viewed with a Zeiss LSM 710 confocal laser-scanning microscope, and representative areas of the sections were photographed under a 40× objective.