Western Blot Analysis of Autophagy Markers

Embryos were deyolked^{80 (link)}, and then homogenized in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Triton X-100, protease inhibitor cocktail and phosphatase inhibitor (Roche)). Protein lysates were separated on SDS-PAGE, transferred to nitrocellulose membrane (Amersham Biosciences), and incubated with anti-LC3 (Cell Signaling Technology, 1:1 000), phos-S6K (Cell Signaling Technology, 1:1 000), phos-RPS6 (Cell Signaling Technology, 1:1 000), phos-elf2a (Cell Signaling Technology, 1:1 000), p62 (MBL,1:1 000), Beclin1 (abcam, 1:5 000), p53 (a gift from Jinrong Peng, Zhejiang University) or anti-α-tubulin (Sigma, 1:2 000) antibodies, and then HRP-conjugated secondary Antibodies.

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Jia X.E., Ma K., Xu T., Gao L., Wu S., Fu C., Zhang W., Wang Z., Liu K., Dong M., Jing C., Ren C., Dong Z., Chen Y., Jin Y., Huang Q., Chang X., Deng M., Li L., Luo L., Zhu J., Dang Y., Chang H.C., Zon L.I., Zhou Y., Chen S, & Pan W. (2015). Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction. Cell Research, 25(8), 946-962.

Publication 2015

nitrocellulose membrane anti-LC3 phos-S6K phos-RPS6 Beclin1 anti-α-tubulin

Antibodies Beclin1 Buffer Edta Embryos Glycerol Membrane Nacl Nitrocellulose Phosphatase Protease inhibitor Protein Sds page Tris Triton x 100 α tubulin

Corresponding Organization :

Other organizations : Shanghai Institutes for Biological Sciences, Shanghai Jiao Tong University, Chinese Academy of Sciences, Fudan University, Shanghai Institute of Hematology, Ruijin Hospital, Southwest University, Boston Children's Hospital, Harvard University

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Variable analysis

independent variables

Deyolking of embryos

dependent variables

Protein expression levels of LC3, phos-S6K, phos-RPS6, phos-elf2a, p62, Beclin1, p53

control variables

Lysis buffer composition (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Triton X-100, protease inhibitor cocktail and phosphatase inhibitor (Roche))
SDS-PAGE separation and nitrocellulose membrane transfer
Antibodies used (anti-LC3, phos-S6K, phos-RPS6, phos-elf2a, p62, Beclin1, p53, anti-α-tubulin)
HRP-conjugated secondary antibodies

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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