Immunoprecipitation buffer

Cells were lysed in immunoprecipitation buffer (Beyotime Biotechnology, Shanghai, China), and equal amounts of 500 μg protein were incubated with specific antibodies against PlexinB1 (ab90087, Abcam), N‐cadherin (ab18203, Abcam), and VE‐cadherin (ab33168, Abcam) overnight at 4°C with gentle rotation. 40 μl Protein A&G Agarose beads (Beyotime Biotechnology) were then added and incubated for another 2 h. Agarose beads were precipitated to bottom by transient centrifugation at 4°C and then washed three times with the lysis buffer. The complexes were eluted from the beads by heating samples in loading buffer with SDS and then used for Western blotting.

Cells were washed 3 times with PBS and resuspended in immunoprecipitation buffer (Beyotime Biotechnology) in the presence of a protease inhibitor cocktail, then were disrupted by sonication, and clarified by centrifugation at 6000 rpm for 10 min. Cell lysates (0.5 mg) were precleared with 20 μl of protein A/G-Sepharose beads (Santa Cruz) for 60 min. Nonspecific complexes were pelleted by centrifugation at 10,000×g at 4 °C for 10 min. The supernatants were removed and incubated with either 2.5 μg of anti-Flag antibody, anti-GFP antibody, or the isotype control IgG at 4 °C for 12 h before the addition of 20 μl of protein A/G-Sepharose beads, and incubated for another 4 h in an end-over-end rotor. The immunoprecipitates were pelleted and washed five times with a lysis buffer. After the final wash, the pellet was resuspended in 40 μl of 2× SDS-PAGE loading buffer and boiled for 10 min before being analyzed by Western blotting with appropriate primary and secondary antibodies.

Protein extracted from complete homogenization of cells in immunoprecipitation buffer (Beyotime, Shanghai, China) was conducted according to the manufacturer's instructions. Protein was separated via SDS–PAGE, and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (MDBio Inc., Shandong, China).
Separated protein blots were incubated at 4°C overnight with 10 ml 5% skim milk powder in Tris‐buffered saline with Tween‐20 (TBST). An anti‐FSTL3 protein antibody (Abcam, Cambridge, UK, 1:2000) was incubated with PVDF membranes at room temperature for 1 hr, followed by washing with TBST three times, 10 min each. Then, diluted Goat anti‐Rabbit IgG (horseradish peroxidase labelled, HRP, Abcam, UK, 1:5,000) was incubated at room temperature for 1 hr, followed by washing in TBST three times, 10 min each. Stock solutions of 20 × LumiGLO and 20 × hydrogen peroxide were diluted to 1 × with ddH₂O, which was added dropwise to the PVDF membranes and incubated in the dark for 1 min, taking images via the Molecular Imager^® Gel Doc™ XR System (BIO‐RAD, USA) in 30 min.

At 24 h after OGD/R, neurons were lysed on ice in immunoprecipitation buffer (Beyotime Institute of Biotechnology) with a protease inhibitor cocktail (Roche, Basel, Switzerland). One-fifth of the cell lysates were prepared as input samples, and the rest were used for coimmunoprecipitation. Cell lysates were pre-cleared with Protein A Sepharose beads (GE Healthcare, Uppsala, Sweden) for 1 h, and the supernatant was incubated with a primary antibody of GRP78 (Abcam, LA, USA) at 4 °C overnight. The Protein A Sepharose beads were then added to the system and incubated for 2 h at 4 °C. After incubation, the beads were washed 3 times with cold PBS. The immunoprecipitates were subjected to Western blotting analysis with an anti-PERK antibody or anti-IRE1 antibody (Abcam, LA, USA).

Renal tissues were ground in immunoprecipitation buffer (Beyotime) supplemented with protease inhibitors and then centrifuged at 8000 g for 10 mins to obtain whole cell lysates. Lysates were immunoprecipitated with the Tet1 antibody or respective IgG with Protein A/G magnetic beads (MedChem Express, NJ) overnight at 4 °C 32 (link). After washing, the beads were boiled in loading buffer and subjected to Western blots.

Total protein extracts were prepared from the spinal cord at the L3-5 segments (4 h after plantar incision or sham operation) using ice-cold immunoprecipitation buffer (Beyotime) containing 0.1 mM phenylmethylsulfonyl fluoride protease inhibitor and a protease inhibitor cocktail (0.02%, v/v; CST). The homogenized samples were centrifuged at 12000 g for 10 min at 4°C. After incubating with rabbit anti-pmTOR (10 μg, Abcam, RRID:AB_10888105) overnight at 4°C, the protein extracts (500 μg) were incubated with protein A/G agarose (Calbiochem, IP05) on a rotator for 3 h. In the negative control group, normal rabbit IgG (2 μg, CST, RRID:AB_1031062) was used in place of anti-pmTOR. After washing with immunoprecipitation buffer five times, the mixture was dissolved in 2× sample buffer and denatured at 95°C for 5 min. Samples containing equal weights of protein (30 μg) or total protein extract (10 μg, as a positive control, input) were detected by western blotting.