Rabbit anti p ire1α

Sciatic nerves and RSC96 cells were lysed on ice in the RIPA buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail for 30 min to extract total proteins. The proteins were analyzed with a bicinchoninic acid (BCA) protein assay kit (Biosynthesis, China). 30 μg/lane (sciatic nerve) and 20 μg/lane (RSC96 cell) were used for Western blot analysis as previously described^{8 (link),27 (link)}. The primary antibodies were as follows: mouse anti-IRE1α (sc-390960; 1: 1000; Santa Cruz), rabbit anti-P-IRE1α (ab48187; 1: 2000; abcam), mouse anti-XBP1 (sc-8015; 1: 1000; Abcam), mouse anti-GRP78 (sc-376768; 1: 1000; Santa Cruz), mouse anti-GADD153 (sc-7351; 1: 500; Santa Cruz), rabbit anti-Caspase-3 (ab13847; 1: 1000; abcam), rabbit anti-Caspase-12 (om273459; 1: 1000; Omnimabs), mouse anti-Bcl-2 (sc-7382; 1: 1000; Santa Cruz), mouse anti-Bax (sc-7480; 1: 1000; Santa Cruz), mouse anti-p-JNK (sc-6254; 1: 1000; Santa Cruz), and rabbit anti-PGP9.5 (ab108986; 1: 2000; Abcam). Mouse anti-β-actin (TA-09; 1: 20000; Zhongshan Goldenbridge) served as the internal control. The secondary antibodies were goat anti-mouse IgG-HRP (ZB-2305; Zhongshan Goldenbridge) and goat anti-rabbit IgG-HRP (ZB-2301; Zhongshan Goldenbridge). Western Chemiluminescent HRP Substrate and exposed to X-film to form image. The protein bands were quantitated with Image J software^{27 (link)}.

The protocol of WB could be referred to the previous studies.^[1, ^39] The total protein was obtained by utilizing the radioimmunoprecipitation assay lysis buffer Beyotime, Jiangsu, China) along with protease and phosphatase inhibitors (BioTools, Olathe, KS, USA), following the instructions provided by the manufacturer.^[40] The protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Bio‐Rad Laboratories, Inc., Berkeley, CA, USA). Equivalent amounts of protein were separated through sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (Beyotime Biotechnology, Shanghai, China) and subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, USA). After blocking with 5% Tris‐buffered saline‐Tween, the membrane was subjected to overnight incubation at 4 °C with the primary antibody. The antibodies utilized in this study were as follows: rabbit anti‐THBS1 antibody (Cell Signaling Technology, Danvers, MA, USA); rabbit anti‐mouse IgG (Thermo Fisher Scientific), rabbit Anti‐ARMET antibody (Abcam), rabbit anti‐Thrombospondin 1 antibody (Abcam), rabbit Anti‐p‐PERK (Thermo Fisher Scientific), rabbit Anti‐PERK (Thermo Fisher Scientific), rabbit Anti‐IRE1α (Cell Signaling Technology, Danvers, MA, USA), rabbit Anti‐p‐IRE1α (Abcam).

The slices were rinsed with PBS for 30 min and permeabilized with 0.3% Triton X-100 for 30 min at room temperature [37 ]. The slices were then rinsed with PBS for 15 min and blocked with 5% donkey serum at 37 °C for 30 min. Subsequently, each coronal slice was incubated with primary antibodies at 4 °C overnight. The primary antibodies used are as follows: rabbit anti-p-IRE1α (1:50, Abcam), mouse anti-NeuN (1:200, Santa Cruz Biotechnology), mouse anti-GFAP (1:100, Santa Cruze Biotechnology), and mouse anti-Iba-1(1: 200, Wako). Slices were rinsed in PBS for 15 min and then incubated with appropriate fluorescence-conjugated secondary antibodies at 37 °C for 1 h. After being rinsed in PBS for 15 min, the slices were covered with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories Inc.). Images were then visualized under a fluorescence microscope (Leica DMi8, Leica Microsystems).