Immunoprecipitation and Western Blot Analysis

For immunoprecipitation, cells were lysed with NP40 lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM DTT, 10% glycerol) containing protease inhibitors. After centrifugation, the supernatants were incubated with antibody overnight and then Protein A/G agarose for 2 h at 4 °C. Immunocomplexes were washed and analyzed by Western blot. For Western blot, the proteins from lysed cells were denatured and separated with SDS-PAGE. Then, the proteins were transferred to PVDF membranes, blocked and incubated with the corresponding primary and secondary antibodies. The specific bands were analyzed by the Western blot infrared imaging system (LI-COR Biosciences). The following antibodies were used: EVA1A: A8070, ABclonal; CHOP: 2895, Cell signaling Technology (CST); IRE1: 14C10, CST; ATF6: 65880 T, CST; Actin: AC026, ABclonal; Flag: 20543-1-AP, Proteintech; Caspase 8: 4927, CST; C-Caspase 9: 9509, CST; C-Caspase 3: 9664, CST; Caspase 12: 2202, CST; C-Parp: 9548, CST; GFP: ab290, Abcam; MCL1: 94296, CST; Bak: 12105, CST; LC3: 27543,Sigma; P62: 18420-1-AP, Proteintech; BNIP3: ab10433, Abcam; TIM23: 111263-A, Proteintech; TOM20: ab56783, Abcam.

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Liu B., Zhou Y., Wu Q., Fu Y., Zhang X., Wang Z., Yi W., Wang H., Chen Z., Song Z., Xiong W., Qiu Y., He W, & Ju Z. (2023). EVA1A regulates hematopoietic stem cell regeneration via ER-mitochondria mediated apoptosis. Cell Death & Disease, 14(1), 71.

Publication 2023

Western blot infrared imaging system BNIP3 ab10433

Actin Agarose Antibodies Antibody Atf6 Buffer Caspase 12 Caspase 3 Caspase 8 Caspase 9 Centrifugation Chop Edta G protein Glycerol Immunoprecipitation Mcl1 Membranes Nacl Np 40 Protease inhibitors Protein a Protein g Proteins Pvdf Sds page Tris Western blot

Corresponding Organization : Southwest Hospital

Other organizations : Hangzhou Normal University, Zhejiang University, Sir Run Run Shaw Hospital, Hefei National Center for Physical Sciences at Nanoscale, University of Science and Technology of China, Weifang Medical University

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Variable analysis

independent variables

Immunoprecipitation with antibodies

dependent variables

Protein expression levels detected by Western blot
Presence and levels of specific proteins (EVA1A, CHOP, IRE1, ATF6, Actin, Flag, Caspase 8, C-Caspase 9, C-Caspase 3, Caspase 12, C-Parp, GFP, MCL1, Bak, LC3, P62, BNIP3, TIM23, TOM20)

control variables

NP40 lysis buffer composition (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM DTT, 10% glycerol)
Protease inhibitors in the lysis buffer
Incubation conditions (overnight incubation with antibody, 2 h incubation with Protein A/G agarose, 4 °C)
SDS-PAGE for protein separation
PVDF membrane for protein transfer
Blocking conditions
Primary and secondary antibodies used for detection

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