Cell lysates were prepared in RIPA buffer and immunoblotting was performed as previously described (19 (link)). Nuclear fractionation was performed using the REAP cellular fractionation method, as described in (35 (link)). Antibodies used were as follows: SPHK1 (Bethyl Laboratories, Montgomery, TX), SGPL1 and HIF1α (Abcam, Cambridge, MA), HIF2α (Novus Biologicals, Littleton, CO), phospho-AKT (S473), AKT, SOX2, phospho-AMPK (T172), AMPK, c-MYC, OCT-1, PKM1/2, β-Tubulin, Histone H3, cleaved caspase 3, Snail, V5-tag and β-Actin (Cell Signaling Technology, Beverly, MA).
Protocol detail
Immunoblotting and Nuclear Fractionation
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HIF2α
phospho-AKT
phospho-AMPK
c-MYC
OCT-1
PKM1/2
β-Tubulin
Histone H3
cleaved caspase 3
Snail
V5-tag
β-Actin
Actin
Antibodies
Biologicals
Buffer
C myc
Caspase 3
Cell
Cellular fractionation
Fractionation
Hif1α
Histone h3
Novus
Oct 1
Ripa
Snail
Sox2
Tubulin
Corresponding Organization : University of Chicago
Other organizations : Keio University, Duke University, University of Wisconsin–Madison
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Variable analysis
independent variables
- Cell lysates were prepared in RIPA buffer
- Nuclear fractionation was performed using the REAP cellular fractionation method
- Immunoblotting was performed as previously described (19)
- Levels of SPHK1, SGPL1, HIF1α, HIF2α, phospho-AKT (S473), AKT, SOX2, phospho-AMPK (T172), AMPK, c-MYC, OCT-1, PKM1/2, cleaved caspase 3, Snail, and V5-tag were measured
- β-Tubulin, Histone H3, and β-Actin were used as loading controls
- Positive control: Not specified
- Negative control: Not specified
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