Goat anti rabbit igg

The protein concentrations of the cell lysate were determined using the Bradford protein assay kit using the bovine serum albumin as the protein standard (Beyotime). Equal amounts of protein (25 μg) per sample were separated on 10 % SDS polyacrylamide gels and then transferred onto 0·45 μm PVDF membranes (Millipore). The membranes were blocked with a blocking buffer (Beyotime) at room temperature for 2 h, followed by 4 h incubation at room temperature with the primary antibodies mentioned in online Supplementary Table S1. After washing with (TRIS-buffered saline containing 0·02 % (v/v) Tween-20 (TBST) three times, the membranes were then incubated with secondary goat-anti-rabbit IgG (Beyotime, dilution 1:500) or goat-anti-mouse IgG (Beyotime, dilution 1:500) conjugated with horseradish peroxidase in a blocking buffer for 4 h at 37°C. The membranes were then washed with TBST three times and immediately incubated with enhanced chemiluminescence Western blotting substrate kits (Beyotime), followed by visualisation using a chemiluminescence imaging system (CLiNX Science Instrument). The relative intensities of the bands were calculated with ImagePro Plus 6.0 software (Media Cybernetics). The β-actin protein was used as the reference protein.

Approximately 1 × 10⁷ cells were solubilized in lysis buffer purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) was utilized to separate the proteins. Afterwards, approximately 60 µg of the purified proteins was transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membrane with the adsorbed proteins was incubated with Tris‐buffered saline with Tween 20 (TBST) buffer obtained from Fanke Biotech Co., Ltd. (Shanghai, China) at room temperature, supplemented with 5% non‐fat milk. After 1 hour, the membrane was incubated with the primary antibodies overnight at room temperature, followed by incubation with the corresponding secondary antibody for 4 hour. In the present study, the primary antibodies used were as follows: rabbit anti‐α‐SMA (ab124964, 1:10 000 dilution; Abcam), rabbit anti‐ITGB3 (ab218435, 1:5000 dilution), rabbit anti‐PARP‐1 (1:1000; Cell Signaling) and γH2AX (1:250; Cell Signaling). The secondary antibody was goat anti‐rabbit IgG (1:5000 dilution; Beyotime) labelled with horseradish peroxidase (HRP). An electrochemiluminescence (ECL) kit and ImageJ software from Media Cybernetics (Rockville, MD, USA) were used to determine the chemiluminescent and relative protein expression, which was presented as the density ratio compared to GAPDH.

Western blot analysis was performed as detailed elsewhere (39 (link)). Twenty micrograms of total proteins obtained from various cell lines were electrophoresed through 10% bis–Tris SDS-polyacrylamide gels. Afterward, the gels were electroblotted onto polyvinylidene difluoride (PVDF) membrane for hybridization. The antibodies used for this investigation were from Invitrogen [TRUB1 (PA5-36003) and ND4L(PA5-68242)], Abcam [ND1(ab74257), ND3(ab170681), ND5(ab92624), TOMM20/TOM20(ab56783), SDHB(ab14714), UQCRC2(ab14745) and TUBULIN (ab6046)], Novus [ND4(NBP2-47365)], ABclonal [NDUFA1(A20940) and NDUFA10(A10123)] and Proteintech [FLAG (80010-1-RR), NDUFS1(12444-1-AP), UQCRFS1(18443-1-AP), COXIV(66110-1-Ig), COX17 (11464-1-AP), CYTB (55090-1AP), CO2 (55070-1-AP), ATP8 (26723-1-AP), ATP5B (17247-1-AP), ATP5F1(15999-1-AP) and GAPDH (60004-1-Ig)]. Peroxidase Affinipure goat anti-mouse IgG and goat anti-rabbit IgG (Beyotime) were used as secondary antibodies, and protein signals were detected using the ECL system (CWBIO). The quantification of density in each band was performed as detailed previously (38 (link)).

After culture, NP cells seeded on the glass coverslips were fixed with 4% paraformaldehyde for 20 min. Then, they were permeabilized with 0.1% Triton X-100 for 30 s, blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies (aggrecan: Novus, NB600-504; collagen II: Abcam, ab185430) overnight at 4°C. After incubation with goat antimouse IgG or goat anti-rabbit IgG (1:400 dilution, Beyotime, China) for 2 h, positive staining was developed and the cellular nucleus was stained with the hematoxylin solution. Finally, staining intensity was analyzed using the Image-Pro Plus software (Version 5.1, Media Cybernetics, Inc.).

The protein concentration of hippocampus and prefrontal cortex was determined by a BCA kit (P00125, Shanghai Beyotime Biotechnology Co., Ltd, China). Samples containing 30 μg of protein were mixed with loading buffer and denatured in boiling water for 10 min. The proteins were separated by 12.5% SDS‐PAGE gels and then eletrically transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, USA). After that, membranes were immersed into TBST solution (TBS containing 0.1% Tween‐20) with 5% bovine serum albumin (BSA) and blocked for 1 h at room temperature. Then membranes were incubated in different primary antibodies (anti‐tubulin: 1/1000, #5568, CST, USA; anti‐Cx36: 1/1000, sc‐398063, Santa Cruz, USA) overnight at 4°C. On the next day, membranes were washed with TBST solution for three times and incubated in appropriate secondary antibodies (goat anti‐rabbit IgG: 1/1000, A0208; goat anti‐mouse: 1/1000, A0216; all from Shanghai Beyotime Biotechnology Co., Ltd, China) for 1 h at room temperature. Finally, bands were detected by Biorad imaging system, and the intensities were analyzed by Image J software.