Horseradish peroxidase hrp conjugated secondary antibody

Protein lysates were derived from tissues and cultured cells using RIPA buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). The protein concentrations were detected using an Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). The lysates were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes (MilliporeSigma, Burlington, MA, USA). The membranes were then blocked in 5% non-fat milk powder in PBS-Tween and incubated with primary antibodies against ARHGAP19 (1:500, sc-398428, Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (1:2000, A3044, ABclonal Technology, Wuhan, China) overnight at 4 °C. After washing with PBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, ABclonal Technology) for 2 h at 37 °C and visualized by chemiluminescent detection using an ECL kit (Beyotime Biotechnology).

Protein lysates were derived from tissues and cultured cells using RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (Beyotime Biotechnology). The protein concentrations were detected using an Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). The absorbance values were detected by using a microplate reader. Protein samples need to be denatured before the experiment. The lysates were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore-Sigma, Burlington, MA, USA). The membranes were then blocked in 5% non-fat milk powder in PBS-Tween and incubated with primary antibodies against ELAC2 (1:500, A7128, ABclonal Technology, Wuhan, China), E-cadherin (1:5000, 20874-1-AP, Proteintech Group, Wuhan, China) and vimentin (1:500, CY5134, Abways Technology, Shanghai, China) overnight at 4 °C. After being washed with PBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, ABclonal Technology) for 2 h at 37 °C and visualized by chemiluminescent detection using an ECL kit (Beyotime Biotechnology). The original pictures of the Western Blot were analyzed by Image J software (1.53t, NIH, Bethesda, MD, USA).

Total proteins were extracted from the cells, and the protein concentration was determined using a BCA assay kit. The proteins were fractionated by using 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto a polyvinylidene fluoride filter membrane (PVDF; Millipore, USA). After incubation with primary antibodies (Wanleibio, China; Abclonal, USA), the PVDF membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abclonal). Finally, the blot was developed using ultrasensitive ECL chemiluminescence reagent (Meilunbio), and the membrane was exposed. The bands were quantified using a chemiluminescence imaging system (Syngene, USA) and analysed using ImageJ.

HLECB3 cells were cultured in a 6-well plate for 24 h. Cells were treated with H₂O₂ (200 μM) or EX-527 (40 nM) or siSirt1 (50 nM), Res (32 μg/mL), and RGNPs (32 μg/mL) for 24 h. Cell lysates were collected and purified using centrifugation at 13,000 rpm at 4 °C for 15 min. Then, cell lysates were mixed with 5x loading buffer (20 μg/lane) and were resolved by electrophoresis on 4–20% prefabricated denaturing polyacrylamide gels (ACE Biotechnology, Xiangtan, China) and transferred to a nitrocellulose membrane. Subsequently, membranes were blocked with 5% fat-free milk and 0.1% Tween 20 in Tris-buffered saline for 1 h at 4 °C, and then incubated with a primary antibody (with 5% fat-free milk above). Further, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:4000) (Abclonal, Wuhan, China). Finally, the membranes were visualized using a ChemiDoc^TM Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).