β-chitosan attenuates hepatic macrophage-driven inflammation and reverses aging-related cognitive impairment

Description

All the target proteins in each picture come from the same piece of gel, and each band is labelled appropriately (including marker), which can be spliced into a complete membrane. Only bands from the same gel are used for data analysis. In addition to a white background photo corresponding to the underlying raw images, we also provide a composite image with marker.

Steps to reproduce

Tissues and cells were lysed and homogenized with RIPA lysis buffer (Beyotime, P0013) containing protease inhibitors (Roche, 04693132001) and phosphatase inhibitors (Roche, 04906 837001). Total protein concentration was determined by BCA assay (ThermoFisher Scientific, 23227). Used 10%-12% SDS-PAGE to process the equal amount of protein and transferred it to PVDF membrane (Roche, 03010040001). The membranes were blocked with 5% no-fat milk/TBST at room temperature for 1 h and then incubated with the primary antibody (1:1000 dissolved in TBST, see key resources table) at 4℃ for at least 17 hours. The secondary antibodies (1:5000, ThermoFisher Scientific, 31234, Rabbit; 31166, Mouse) of the corresponding species were incubated at room temperature for 1 hours after the first antibody was recovered, visualized by using ECL Western Boltting Substrate. The primary antibodies for western blot, mice: VCAM1 (Abcam, ab134047, RRID:AB_2721053), CD68 (ProteinTech, 28058-1-AP), IL-1β (Santa Cruz Biotechnology, SC-52012), TNF-α (Affinity, AF7014), IBA1 (ProteinTech, 10904-1-AP), NLRP3 (Affinity, DF7438), MMP9 (Abmart, TA0220), IL-6 (Abmart, TD6087); cell: iNOS (Affinity, AF0199), CD86(Affinity, DF6332), TLR4 (ProteinTech, 19811-1-AP); JNK (ProteinTech, 24164-1-AP); p38 (ProteinTech, 14064-1-AP); p65 (ProteinTech, 10745-1-AP); p-JNK (Affinity, AF3318); p-p38 (Affinity, AF4001); p-p65 (Affinity, AF2006), TNF-α (Affinity, AF7014), IL-1β (Affinity, AF5103); zebrafish: IL-1β (ProteinTech, 16806-1-AP). β-tubulin (Affinity, AF7018), GAPDH (Affinity, AF7021), β-actin (Affinity, AF7011).

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