mouse liver nuclear lysates  (Santa Cruz Biotechnology)

Journal: Cells

Article Title: OGT ( O -GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A

doi: 10.3390/cells7050044

Figure Lengend Snippet: O -GlcNAcylation of native lamin A in human hepatoma (Huh7) cells and mouse liver. ( A ) Western blots of proteins from human hepatoma (Huh7) cells, cultured for 24 h in physiological (5 mM) or high (25 mM) glucose, probed with antibody CTD 110.6 specific for the β- O -linked N -acetylglucosamine modification ( O -GlcNAc), and then stripped and re-probed with antibody 5G4, specific for lamins A and C (Lamin A/C). Control input lysates, and proteins immunoprecipitated using lamin A/C antibody 5G4 or nonspecific IgG as the negative control, were resolved in parallel gels. Asterisk indicates the O -GlcNAc signal corresponding to immunoprecipitated lamin A; ( B ) Western blots of mouse liver nuclear proteins, before (Input) or after affinity purification on succinylated Wheat Germ Agglutinin (sWGA), which binds O -GlcNAc-modified proteins. We analyzed two livers for each condition. Male mice were either fasted (Fasted), or fasted and then re-fed for 12 h (‘Refed’), and were either pretreated with streptozotocin to induce hyperglycemia (‘STZ’), or not (‘Vehicle’). Input controls were probed with lamin A-specific antibody L1293. Affinity-purified samples were probed first for the O -GlcNAc modification (antibody CTD 110.6 ), and then stripped and re-probed for lamin A (antibody L1293). Control input lysates, and proteins immunoprecipitated using lamin A/C antibody 5G4 or nonspecific IgG as the negative control, were resolved in parallel gels. STZ—streptozocin.

Article Snippet: Mouse liver nuclear lysates (100 μg per sample) were incubated for 1 h (4 °C) with protein A/G agarose beads (sc-2003; Santa Cruz Biotechnology), then centrifuged.

Techniques: Western Blot, Cell Culture, Modification, Control, Immunoprecipitation, Negative Control, Affinity Purification