Immunoprecipitation and Western Blot Analysis

Immunoprecipitation was conducted as previously described ^{62 (link), 63 (link)}. Protein extracts were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad) using standard techniques. The primary antibodies and concentrations used for Western blotting were: BAP1 (1:1000; Santa Cruz, sc-28383), SLC7A11 (1:5000; Cell Signaling Technology, 12691), tubulin (1:5000; Cell Signaling Technology, 2144), H2Aub (1:5000; Millipore, 05–678), H2A (1:5000; Millipore, ABE327), vinculin (1:10000; Sigma, V4505), FOXK1 (1:1000; Abcam, ab18196 ), FOXK2 (1:1000; Bethyl Laboratories, A301–730A), HCFC1 (1:1000; Bethyl Laboratories, A301–399A), OGT (1:1000, Cell Signaling Technology, 5368), KDM1B (1:1000; Abcam, ab193080), ASXL1 (1:1000; Santa Cruz, sc-293204), p53 (1:1000; Santa Cruz, sc-126), PARP (1:1000; Cell Signaling Technology, 9542), β-actin (1:4000; Abcam, ab8226), and V5 (1:2000; Sigma, A8012).

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Zhang Y., Shi J., Liu X., Feng L., Gong Z., Koppula P., Sirohi K., Li X., Wei Y., Lee H., Zhuang L., Chen G., Xiao Z.D., Hung M.C., Chen J., Huang P., Li W, & Gan B. (2018). BAP1 links metabolic regulation of ferroptosis to tumor suppression. Nature cell biology, 20(10), 1181-1192.

Publication 2018

nitrocellulose membrane sc-28383 SLC7A11 tubulin H2Aub V4505 FOXK1 FOXK2 HCFC1 KDM1B ASXL1 sc-293204 β-actin ab8226

A301 Actin Antibodies Immunoprecipitation Membrane Nitrocellulose Parp 1 Protein Sds page Tubulin Vinculin

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, Baylor College of Medicine, The University of Texas Health Science Center at Houston

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Variable analysis

independent variables

Immunoprecipitation protocol

dependent variables

Protein expression levels of BAP1, SLC7A11, tubulin, H2Aub, H2A, vinculin, FOXK1, FOXK2, HCFC1, OGT, KDM1B, ASXL1, p53, PARP, β-actin, and V5

control variables

Standard techniques for SDS-PAGE and Western blotting

controls

Positive controls: None specified
Negative controls: None specified

Annotations

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