Hrp conjugated goat anti mouse igg h l

Mouse kidney samples were treated with radioimmunoprecipitation assay (RIPA) buffer for protein extraction. Protein concentration was subsequently determined using a BCA kit. The samples were denatured for Western blotting. Briefly, protein samples were loaded into the gel, and sodium dodecyl benzene sulfonate gel electrophoresis was performed for 1–2 h. Proteins were then transferred to the PVDF membrane by the wet method, blocked for 1 h in a 3% TBS-T skim milk solution, and incubated overnight in the primary antibody solution at 4 °C. The next day, the membranes were washed three times with standard TBS-T buffer, incubated for 2 h at room temperature in a secondary antibody solution, washed three times, and incubated with ECL prior to detection in an imaging system. Images were analyzed in the "ImageJ" software. The mouse anti-GAPDH antibody was purchased from TransGen Biotech Co., Ltd. (Beijing, China). HRP-conjugated Goat Anti-Mouse IgG (H + L) and HRP-conjugated Goat Anti-Rabbit IgG (H + L) were purchased from Servicebio Technology Co., Ltd. (Wuhan, China). Mouse Anti-Nrf2 was acquired from Proteintech Group Inc. (Chicago, IL, USA), and Rabbit Anti-HMGB1 and Rabbit Anti-TLR4 antibodies were purchased from Affinity Biosciences Ltd. (Melbourne, Australia).

Treated LUAD cells were extracted by RIPA lysis buffer (C1053, Beijing Pulilai Gene Technology Co., Ltd., China). The protein concentrations were measured by BCA method (E-BC-K318-M, Elabscience, China). 40 μg of protein was separated by 10% SDS-polyacrylamide gels. The protein samples were transferred onto PVDF membranes (IPVH00010, Millipore, USA). The membranes were blocked with 5% BSA for 2 h. Followed by incubation with primary antibodies, including Rabbit Anti-DARS2 (DF12593, Affinity, China, 1/1000) and Mouse Anti-β-Actin (HC201, TransGen Biotech, China, 1/2000). The membranes were washed with TBST, and then incubated with corresponding secondary antibodies, including HRP conjugated Goat Anti-Mouse IgG (H+L) (GB23301, Servicebio, China, 1/2000) and HRP conjugated Goat Anti-Rabbit IgG (H+L) (GB23303, Servicebio, China, 1/2000), at room temperature for 1 h. Finally, the proteins were visualized by an enhanced chemiluminescence (ECL) detection kit (RJ239676, Thermo Fisher Scientific, Inc., USA). We captured the chemiluminescent signal of protein bands using the Tanon-5200 chemiluminescence imaging system (Tanon, Shanghai, China). The optical density of protein bands was quantified by using ImageJ software (Image J Software Inc., USA).