Pb0981

Immunohistochemistry for the protein expression of mouse kidneys was assessed using the streptavidin–peroxidase immunohistochemical method. For immunohistochemistry staining, deparaffinized sections were boiled in citrate buffer for antigen retrieval. Then, the sections were permeabilized in 1× PBS containing Triton X-100 (0.1%) for 10 min and incubated with anti-α smooth muscle actin (α-SMA, GB11044, Servicebio, Wuhan, China), antifibronectin (GB112093, Servicebio), anticollagen I (PB0981, BOSTER, Wuhan, China), anti-PKM2 antibody (4053S, CST, Boston, United States), anti-p-PKM2 (PA5-105498, Invitrogen, California, United States), anti-CD206 (ab125028, Abcam, Cambridge, United Kingdom), anti-inducible nitric oxide synthase (iNOS, ab178945, Abcam) at 4°C overnight. Immunohistochemical staining was performed using an UltraSensitive S-P kit (kit 9706, Maixin-Bio, Jinan, China). The selected regions were captured under a light microscope.

The samples were decalcified in 10% disodium ethylenediaminetetraacetic acid (Solarbio, China) at 4 °C for 30 d. Afterwards, decalcified samples were longitudinally embedded in paraffin wax and sliced into 5 μm sections. Then, the tissue sections were stained with haematoxylin and eosin (Solarbio) and TRAP staining kit (Solarbio) based on the manufacturer’s instructions. The stained sections were photographed under the microscope (DM6000 B, Leica) and analyzed with Image ProPlus 6.0 Software (Media Cybernetics, Silver Spring, USA). OCN (PB1009, Boster) and COL-1a (PB0981, Boster) staining was performed with an anti-rabbit HRP/DAB Detection Kit (18653 s, CST), following the manufacturer’s protocol. The images were photographed under the microscope. Semiquantitative analysis was measured by Image ProPlus 6.0 Software. For histology evaluation, 3 experienced orthopedical pathologists were invited.

LX-2 cells were rinsed twice with ice-cold PBS and then lysed in RIPA lysis buffer (Sigma-Aldrich). The lysed cells were centrifuged at 10,000 g for 15 min at 4°C, and the supernatants were harvested. The protein concentration of each supernatant fraction was determined using a BCA kit. Equal amounts of protein (40 μg) were separated by 12% SDS-PAGE, and the protein bands were transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk in TBST and subsequently incubated with primary antibodies against α-SMA (Servicebio, GB111364, 1 : 800), ASK1 (Boster Bio, A00929, 1 : 1000), p-ASK1 (Cell Signaling Technology, #3765, 1 : 1000), Col I (Boster Bio, PB0981, 1 : 1000), Col III (Boster Bio, M00788, 1 : 1500), GRP78 (Boster Bio, PB0669, 1 : 1200), PERK (Boster Bio, A01992-2, 1 : 1500), p-PERK (Cell Signaling Technology, #3179, 1 : 1000), CHOP (Abcam, ab179823, 1 : 2000), CD63 (Boster Bio, PB9250, 1 : 1500), TSG101 (Boster Bio, PB0550, 1 : 1000), and GAPDH (Boster Bio, BM1623, 1 : 1000) overnight at 4°C. The next morning, the membranes were washed with TBST and then incubated with the HRP-conjugated secondary antibody for 2 h at room temperature. The immunostained protein bands were visualized by enhanced chemiluminescence, with GAPDH serving as an internal control.