D110016

Protein samples from AD and normal tissues were added to sodium dodecyl sulfate-polyacrylamide gel (15 μg sample per gel lane) to separate, and then, the protein bands were transferred to polyvinylidene fluoride membranes. The membranes were blocked, followed by incubation with rabbit anti-YTHDC1 (abs117811, Absin, Shanghai, China) and rabbit anti-GAPDH (D110016, Sangon Biotech, Shanghai, China). The blots were then incubated with goat anti-rabbit IgG which was labeled by horseradish peroxidase. Finally, the density of immunoreactive bands normalized to the signal intensity of GAPDH was determined using the Electro Chemical Luminescence Kit and ImageJ software 1.53.

The total protein extracted from cells were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and then transferred to a PVDF membrane. The membrane was next incubated with 5% skim milk for 2 h at 20-25°C. After being incubated with primary antibodies at 4°C for 10h and then with rabbit or mouse secondary antibodies at 20-25°C for 1h. Signals were detected using chemiluminescence reagents. The primary antibodies used were anti‐XBP1 (1:1000, ab37152, abcam), anti‐IGFBP3. (1:1000 10189-2-AP,PTGCN), anti‐Tubulin antibody (1:5000. D110016, Sangon Biotech, Shanghai, China).

Cells were transfected with the circ_0075062 interference plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. Then, transfected cells were collected for western blotting analysis after 24 h of glucose deprivation incubation. Briefly, treated NP cells were completely lysed in ice-cold RIPA Lysis Buffer containing 1% Protease Inhibitor Cocktail (Sigma-Aldrich) and then centrifuged at 12000 r/min for 15 min. The bicinchoninic acid protein assay was performed to monitor the protein concentration. Then, proteins were separated in a 10% SDS-PAGE gel and transferred to PVDF membranes. The membranes were subsequently blocked with 3% bovine serum albumin/Tris-buffered saline containing Tween 20 for 2 hours and then incubated with anti-MMP-13 (Affinity, AF5355, 1 : 1000), anti-aggrecan (Affinity, DF7561, 1 : 1000), anti-collagen II (Affinity, AF0135, 1 : 1000), and anti-GAPDH (Sangon, D110016, 1 : 1000) at 4°C overnight and then incubated with a 1 : 5000 secondary antibody for 2 hours. The protein signals were detected by the enhanced chemiluminescence method. GAPDH was used as an endogenous normalization of protein. The intensities of bands were analyzed using ImageJ.

Briefly, the adherent cell culture was washed with PBS on ice. Then, whole-cell lysates were prepared via cell lysis using RIPA Lysis Buffer (P0013B, Beyotime Biotech, Shanghai, China) containing 1X Protease and phosphatase inhibitor cocktail (P1045, Beyotime Biotech, Shanghai, China) and subjected to ultrasonication. The total protein concentration in the lysate was determined using the BCA Protein Assay Kit (P0012, Beyotime Biotech, Shanghai, China). Then, 20 μg of proteins was subjected to 10% SDS-PAGE. The separated protein bands were immunoblotted onto polyvinylidene difluoride membranes (ISEQ 000 10, Millipore, Burlington, MA, USA). The membranes were blocked with 5% non-fat milk powder in TBS buffer. Primary antibodies were incubated overnight at 4 °C followed by secondary antibodies for 2 h at room temperature. After adding Super Signal West Pico PLUS substrate (Cat 34580, ThermoFisher Scientific, Waltham, MA USA), chemiluminescence signals on the membrane were visualized using iBright FL1000 (ThermoFisher Scientific, Waltham, MA USA). The antibodies used in this study were anti-PLK1 rabbit polyclonal antibody (1:500 dilute; D321829, Sangon Biotech, Shanghai, China), anti-GAPDH rabbit polyclonal antibody (1:6000 dilute; D110016, Sangon Biotech, Shanghai, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, Shanghai, China).