Horseradish peroxidase conjugated goat anti rabbit antibody

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.

The specific experimental methods are similar to those previously described [4 (link)]. Briefly, 200 μg exosomes or 3 × 10⁵ TSC-Exos-treated tenocytes per group were lysed with RIPA buffer (Beyotime, Shanghai, China). The protein concentration of the lysate was estimated using the bicinchoninic acid assay (Beyotime, Shanghai, China). For each sample, 20 μg of total protein was used for polyacrylamide gel electrophoresis. After that, the protein was transferred to polyvinylidene difluoride (PVDF) membranes. The primary antibodies used in this study were Rabbit monoclonal anti-CD9 (1:2000; Abcam), anti-TSG101 (1:2000; Abcam), anti-Hsp70 (1:1000; Abcam), anti-AKT (1:1000; Cell Signaling Technology), anti-phospho-AKT (1:2000; Cell Signaling Technology), anti-ERK1/2 (1:1000; Cell Signaling Technology), and anti-phospho-ERK1/2 (1:2000; Cell Signaling Technology). Horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5000; Boster, China) was used as the secondary antibody. The test was repeated three times. ImageJ software was used to analyze the gray value of the final protein band.

ADSC lysis was performed in RIPA buffer (Beyotime) for protein extraction. The protein concentrations in the lysates were estimated by performing dipicolinic acid assays (Beyotime). Immunoblotting was performed with primary rabbit antibodies against TSG101 (monoclonal; 1 : 2000; ab125011; Abcam), CD9 (monoclonal, 1 : 2000; ab92726; Abcam), Hsp70 (monoclonal; 1 : 1000; ab2787; Abcam), ERK1/2 (1 : 1000; Cell Signaling Technology), phospho-ERK1/2 (1 : 2000; Cell Signaling Technology), Akt (1 : 1000; Cell Signaling Technology), and phospho-Akt (1 : 2000; Cell Signaling Technology). A horseradish peroxidase-conjugated goat anti-rabbit antibody (1 : 5000; Boster, China) was employed as the secondary antibody. ImageJ software was used for 4densitometric analysis of the final protein bands.