Whole cell lysates were collected in Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40). Isolation of cytosolic and nuclear fractions was done with an NE-PER kit (Pierce; Rockford, IL) following the manufacturer’s protocol. Protein (30 μg) was resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted overnight at 4 °C with primary antibodies. The various primary antibodies used were CTD110.6 (IgM, UAB), GLI1 (Cell Signaling; 2643), GLI2 (Boster Biological Technology; Pleasanton, CA; PA1941), Histone H3 (Cell Signaling; 4499), OGA (Santa Cruz Biotechnology; Dallas, TX; sc-376429), OGT (Sigma-Aldrich; O6264), PKM2 (Cell Signaling; 4053), phospho-Y105–PKM2 (Cell Signaling; 3827), and RL2 (Abcam; Cambridge, MA; ab2739). Alpha-tubulin (Cell Signaling; 12351) or beta-actin (Sigma-Aldrich; A3854) was used to confirm equal loading. Anti-rabbit or anti-mouse HRP-conjugated secondary antibody (GE Healthcare; Chicago, IL) was used for detection, and blots were developed with either ECL or SuperSignal substrate (Pierce) and imaged on films or Amersham Imager 600. The purity of cytosolic and nuclear fractions was confirmed with beta-tubulin (2146; Cell Signaling) or histone deacetylase 1 (Cell Signaling; 2062) antibodies, respectively. The depicted images are representative of experiments repeated at least three times.
Histone deacetylase 1
Manufactured by Cell Signaling Technology
Histone deacetylase 1 is an enzyme that catalyzes the removal of acetyl groups from lysine residues on histone proteins. It is involved in the regulation of gene expression by modulating chromatin structure.
Automatically generated - may contain errors
Lab products found in correlation
β tubulin, by Cell Signaling Technology (2 mentions)
Phospho y105 pkm2, by Cell Signaling Technology (2 mentions)
Anti rabbit or anti mouse hrp conjugated secondary antibody, by GE Healthcare (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Amersham imager 600, by Cytiva (2 mentions)
β actin, by Merck Group (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
2 protocols using histone deacetylase 1
1
Subcellular Fractionation and Immunoblotting
2
Subcellular Fractionation and Immunoblotting
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Whole cell lysates were collected in Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40). Isolation of cytosolic and nuclear fractions was done with an NE-PER kit (Pierce; Rockford, IL) following the manufacturer’s protocol. Protein (30 μg) was resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted overnight at 4 °C with primary antibodies. The various primary antibodies used were CTD110.6 (IgM, UAB), GLI1 (Cell Signaling; 2643), GLI2 (Boster Biological Technology; Pleasanton, CA; PA1941), Histone H3 (Cell Signaling; 4499), OGA (Santa Cruz Biotechnology; Dallas, TX; sc-376429), OGT (Sigma-Aldrich; O6264), PKM2 (Cell Signaling; 4053), phospho-Y105–PKM2 (Cell Signaling; 3827), and RL2 (Abcam; Cambridge, MA; ab2739). Alpha-tubulin (Cell Signaling; 12351) or beta-actin (Sigma-Aldrich; A3854) was used to confirm equal loading. Anti-rabbit or anti-mouse HRP-conjugated secondary antibody (GE Healthcare; Chicago, IL) was used for detection, and blots were developed with either ECL or SuperSignal substrate (Pierce) and imaged on films or Amersham Imager 600. The purity of cytosolic and nuclear fractions was confirmed with beta-tubulin (2146; Cell Signaling) or histone deacetylase 1 (Cell Signaling; 2062) antibodies, respectively. The depicted images are representative of experiments repeated at least three times.
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