RIPA buffer supplemented with 1% PMSF and protease inhibitor cocktail was used to extract protein from both cell lines and tissues. The protein concentrations were measured using BCA assay (Beyotime). Different Samples were separated on 6%–15% SDS‐PAGE gels, followed by protein transfer onto the PVDF membrane (Millipore, USA). After treatment with 5% nonfat milk, the membranes were probed with diluted primary antibodies against β‐Actin (#8457, CST), GAPDH (#5174, CST), β‐Tubulin (#2146, CST), TAOK3 (#ab70297, Abcam), KMT2C (#28437‐1‐AP, Proteintech), ETV5 (#ab102010, Abcam), IRGM (#AP11128b, Abcepta), H3K4me3 (#9727, CST), LC3B (#ab192890, Abcam), P62 (#ab109012, Abcam), FLAG tag (#66008‐4‐lg, Proteintech), Cleaved PARP (#5625, CST), Caspase‐3 (#A2156, Abclonal), BCL‐2 (#A19693, Abclonal), BAX (#41162, CST), BAK (#A10754, Abclonal), Lamin B (#A11459, Abclonal), P‐KMT2C‐S4588 (#E25915, generated from Abclonal) and P‐KMT2C‐T4589 (#E25804, generated from Abclonal) overnight at 4 °C. After the membranes were washed three times with TBST for 10min each, the membranes were incubated by corresponding HRP‐labeled secondary antibody for 2h at room temperature. Signals were examined using an ECL detection system (Tanon).
Lamin b
Manufactured by ABclonal
Sourced in United States
Lamin B is a nuclear envelope protein that is a structural component of the cell nucleus. It plays a crucial role in maintaining the structural integrity and organization of the cell nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bcl 2, by ABclonal (2 mentions)
Bca assay, by Beyotime (1 mentions)
Flag tag (flag), by Proteintech (1 mentions)
Caspase 3, by ABclonal (1 mentions)
Odyssey infrared imaging, by LI COR (1 mentions)
Cleaved caspase 3, by ABclonal (1 mentions)
Gapdh, by ABclonal (1 mentions)
Bcl2 associated x (bax), by ABclonal (1 mentions)
2 protocols using lamin b
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Apoptosis and Oxidative Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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We lysed PIG1 cells and performed a total protein extraction. We quantified the protein content of the samples using a BCA Protein Assay Kit. We chose 10% or 12% SDS-PAGE gels according to the specific target protein sizes. We loaded 30 μg protein samples onto each well and separated the proteins by electrophoresis. The separated proteins were transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking in 5% nonfat milk diluted in TBST for 1 h, we incubated the membranes with the primary antibodies overnight at 4 °C. The primary antibodies used in the experiment were the following: Bax, Bcl-2, Cleaved Caspase-3, GAPDH, Lamin B (ABclonal Biotechnology, USA), Nrf2 and HO-1 (Proteintech, Wuhan, China). On the following day, we washed the membranes with TBST and then incubated them with fluorescence-conjugated secondary antibodies. Finally, we visualized the target proteins using the Odyssey Infrared Imaging (LI-COR Biosciences, United States) and analyzed the band intensities using the ImageJ software.
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