Anti grp78

H9c2 cells and cardiac tissue were lysed with RIPA including PMSF (Phenylmethanesulfonyl fluoride, Beyotime, Shanghai, China). The concentration of protein was checked by BCA Protein Assay Kit (Beyotime, Shanghai, China). Subsequently, the protein was separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The primary antibodies included: anti-GRP78 (1:1000, proteintech, Wuhan, China), anti-CHOP and anti-F4/80 (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Bcl2 and anti-Bax (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-β actin (1:1000, BOSTER, Wuhan, China), anti-GAPDH (1:1000, Beyotime, Shanghai, China). Secondary antibodies were: HRP-Conjugated AffiniPure Goat Anti-Rabbit IgG and HRP-Conjugated AffiniPure Goat Anti-Mouse IgG (1:2000, ZSGB-BIO, Beijing, China). Immunoreactive proteins were visualized using ECL Western blot detection reagents (Cell Signaling Technology, USA).

Rat Müller cells were washed with PBS and lysed with RIPA lysis buffer (Beyotime, China) on ice for 30 minutes. The protein concentrations were detected by the BCA protein assay (Beyotime). The proteins were separated in 4–6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, China) and then transferred to a PVDF membrane (Beyotime). After blocking with 5% nonfat milk in TBST buffer (200 mM Tris and 1.5 M NaCl with 0.1% Tween 20), the membranes were blotted at 4°C overnight with the following primary antibodies: anti-XBP1s (1 : 600; Proteintech), anti-GRP78 (1 : 800; Proteintech), anti-VEGF (1 : 600; Proteintech); anti-ATF4 (1 : 800; Proteintech), and anti-ATF6 (1 : 200; Proteintech). The membranes were sequentially probed with horseradish peroxidase-conjugated goat anti-rabbit antibody (Boster Biological Technology) at room temperature for 2 hours. β-Actin was served as the loading control. Enhanced chemiluminescence (ECL, Beyotime) was used for imaging, and finally, the optical density of the band was analyzed by GeneTools software (Syngene, Synoptic Ltd. USA). All experiments were repeated for 3 times independently.

Total ovary lysates were separated on SDS-PAGE. After electrophoresis, the protein was transferred to PVDF membrane. The membranes were blocked with 5% skimmed milk and incubated with anti-GRP78 (1:1000, #11587–1-AP, Proteintech, Hubei, China), anti-PARP1 (1:4000, #66520-1-lg, Proteintech), anti-PAR (1:1000, #4335-MC-100, Bio-techne, Minneapolis, USA), anti-mTOR (1:1000, #20657-1-AP, Proteintech), anti-p-mTOR (1:800, #sc-293133, Santa cruz, Dallas, Texas, USA), anti-KitL (1:1000, #bs-0545R, Biorbyt, Hubei, China), anti-p-IRE1α (1:800#, bs-16698R, Biorbyt), anti-IRE1α (1:1000, #27528-1-AP, Proteintech), anti-ATF4 (1:1000, #10835-1-AP, Proteintech), anti-p-PERK (1:800, #Orb336657, Biorbyt), anti-PERK (1:1000, #24390-1-AP, Proteintech), anti-HA-tag (1:1000, #30701ES60, YEASEN), anti-FLAG-tag (1:1000, #30501ES60, YEASEN) and anti-GAPDH (1:4000, #ab8245, Abcam, Cambridge, UK) antibodies overnight at 4 °C. After TBST washing, the membranes were treated with the corresponding peroxidase-conjugated secondary antibody, and then the signal was detected with an Enhanced Chemiluminescence Detection Kit (#32106, Thermo Scientific, Waltham, MA, USA) on Tanon 4500 gel imaging system (Shanghai, China).

Colon sections were incubated with anti‐LC3II (1:100, #2775, CST) and anti‐GRP78 (1:100, 66574‐1‐lg, Protein Tech) antibodies. Secondary antibodies (FITC‐conjugated AffiniPure goat anti‐mouse IgG, BA1101, TRITC‐conjugated AffiniPure goat anti‐rabbit IgG, BA1090, Boster, USA) were used in this study.
Cells were incubated with anti‐LC3II (1:500). Nuclei were stained with DAPI (1155MG010, Biofroxx, Germany). Images were obtained using a LEICA SP8 confocal microscope (LEICA, Germany). And the fluorescence intensity was quantified using ImageJ software (Fiji, ImageJ).

Liver lysates and serum samples were prepared, and Western blotting was performed as previously described (Liu et al., 2014). Proteins were resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight with primary antibody at 4°C. Polyclonal rabbit anti‐LC3B (1:1000, Abcam, ab48394), anti‐p62 (1:1000, Sigma‐Aldrich, P0067), anti‐Atg7 (1:1000, Cell Signaling Technology, 8558), anti‐p16 (1:1000, Proteintech Group, 10883‐1‐AP), anti‐p21 (1:1000, Proteintech Group, 10355‐1‐AP), anti‐GRP78 (1:1000, Proteintech Group, 11587‐1‐AP), anti‐GRP94 (1:1000, Cell Signaling Technology, 2104), and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody (1:20,000; Sigma‐Aldrich, G9545) were used for immunoblotting. Western blotting was visualized on the Kodak Image Station (Carestream Health Inc., Rochester, NY) and analyzed with the ImageJ software.

Western blot was performed as previously described (Wang et al., 2019 (link)). Briefly, whole cell extracts were prepared using lysis buffer (50 mM Tris–HCl, 0.5% (vol/vol) NP-40, 1% Triton-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% protease inhibitor cocktails, 1 mM Na₃VO₄, and 1 mM NaF, pH 7.4). Proteins were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes or PVDF membranes. The membranes were incubated in 0.1% PBST with 5% BSA, followed by PBS washing and primary antibody incubation at 4°C overnight. The primary antibodies included: anti-ZIKV envelope (E) (GeneTex, GTX 133314), anti-p62 (Santa Cruz, sc-28359), anti-GRP78 (Proteintech, 11587-1-AP), anti-Calnexin (Proteintech, 66903-1-lg), anti-FLAG (MBL, PM020), and anti-anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, 10494-1-AP). Detection was performed with IRDye 800 CW-conjugated anti-rabbit IgG and IRDye 680 CW-conjugated anti-mouse IgG secondary antibody (LI-COR) according to the manufacturer’s protocols. Immunoreactive bands were visualized using an Odyssey infrared imaging system (LI-COR) as described previously (Doms et al., 2018 (link)).The western blot bands were quantified by Quantity One (Bio-Rad).