Hrp conjugated igg

Protein extracts were prepared from a different group of MLE-12 cells. The samples were incubated for 2 h with primary antibodies against TLR4 (proteintech, 1:5000), γ-H2AX (Cell Signaling Technology; 1:1000), E-cadherin (Cell Signaling Technology, 1:1000), Vimentin (Cell Signaling Technology, 1:1000), and α-SMA (Cell Signaling Technology, 1:1000) in 5% nonfat milk. The samples were then incubated with HRP-conjugated IgG (proteintech, 1:5000) for 1 h.

Proteins were extracted from cells using RIPA lysis Buffer. The extracted proteins were quantified using the Enhanced BCA Protein Assay Kit (Beyotime), denatured by heat, isolated by SDS‐PAGE electrophoresis, and transferred onto PVDF membranes. After blocking with 1% bovine serum albumin solution for 1 h, the membranes were incubated overnight with primary antibodies (NLRP3: 1:1000, Proteintech; cleaved caspase‐1: 1:1000, Proteintech) at 4°C, followed by incubation with HRP‐conjugated IgG (1:5000; Proteintech). The membranes were exposed using FluorChem M Imaging systems. Relative densitometry was analyzed using Image J2x analysis software (NIH).

Brain samples were collected and proteins were obtained with lysis buffer (Thermo Scientific) supplemented with protease inhibitors (Sigma‐Aldrich). After electrophoresis, the proteins were transferred to PVDF (polyvinylidene fluoride) membranes. The membranes were blocked by incubation with 5% dry milk for 2 h at room temperature and then incubated with primary antibodies against ferritin (diluted 1:1000, Abcam, ab75973), alpha smooth muscle actin (α‐sma, diluted 1:1000, Abcam, ab5694), TAGLN / transgelin (Sm22α, diluted 1:1000, Abcam, ab89989), and GAPDH (diluted 1:2000, zsbio, TA‐08) at 4°C overnight. After thorough washing, the membranes were incubated with HRP‐conjugated IgG (diluted 1:2000, Proteintech) for 2 h at room temperature. The protein bands were visualized using ECL kits (Thermo Scientific) and imaged with a ChemDoc MP imaging system (Bio‐Rad). Images of the blots were analyzed using ImageJ software (NIH).