BMDMs were washed twice with cold PBS and then lysed with Protein Extraction Reagent (CWBIO, China), which contained both protease inhibitor (CWBIO, China) and phosphatase inhibitors (Roche, Switzerland). The protein concentration in lysates was determined by the BCA assay (Thermo Fisher Scientific). Equal amounts of protein of each group were loaded onto SDS-PAGE gels and subsequently transferred to PVDF membranes. Next, membranes were blocked in 10% BSA and incubated with a primary antibody against Arg-1 (1 : 1000, Abcam, USA), iNOS (1 : 1000, Abcam, USA), PI3K (1 : 1000, CST, USA), p-AKT (1 : 1000, CST, USA), AKT (1 : 1000, CST, USA), β-tubulin (1 : 2000, ZSJQ-BIO, China), and GAPDH (1 : 2000, ZSJQ-BIO, China) overnight at 4°C. Then, the membranes were washed with TBST three times and incubated with the corresponding HRP-conjugated secondary antibodies for 1 h at room temperature. The blotting was detected by the ECL detection system (PPLYGEN, China) and analyzed by ImageJ software (NIH, MD). β-Tubulin and GAPDH were used as internal controls.
Protein extraction reagent
Manufactured by CWBIO
Sourced in China
The Protein Extraction Reagent is a laboratory product designed for the extraction and isolation of proteins from various biological samples. It is a versatile tool used in biochemical research and analysis.
Automatically generated - may contain errors
2 protocols using protein extraction reagent
1
Western Blot Analysis of Murine BMDMs
2
Protein Expression Analysis of Testes
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Total protein was extracted from the testes with a protein extraction reagent (CWBio, Beijing, China). Western blotting was carried out, as previously described11 . The membranes were exposed to anti‐VDR (1:100), anti‐PPAR‐γ (1:100), anti‐TGF‐β1 (1:500), anti‐NF‐κB (1:500), anti‐phospho‐NF‐κB (1:500), anti‐B‐cell lymphoma 2 (1:500), anti‐Bax (1:1,000), anti‐inhibitor of NF‐κB alpha (1:500) and anti‐β‐actin (1:1,000). After incubation with the horseradish peroxidase‐conjugated secondary antibody, the membranes were treated with enhanced chemiluminescence substrate (Life Technologies, Carlsbad, California, USA) and exposed to X‐ray film. The relative optical density of each target protein was determined and normalized to β‐actin levels using ImageJ software (public domain; National Institutes of Health, Bethesda, Maryland, USA).
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