Western Blot Analysis of Cellular Protein Modifications

Briefly, MV4-11 cells were incubated
with inhibitors prior to cell lysis with radioimmunoprecipitation
assay (RIPA) buffer (20 mM Tris pH 7.4, 150 mM NaCl, 0.5% deoxycholate,
1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS)). Total protein
content was determined through a BCA assay (ThermoFisher), resolved
via a 4–20% polyacrylamide SDS gel, and transferred to a nitrocellulose
membrane (Bio-Rad). The membranes were blocked with a 5% solution
(skimmed milk powder in PBS-T). This was followed by incubation at
4 °C (overnight) with the following antibodies: acetylated α-tubulin
mouse monoclonal (MABT868, EMD Millipore), acetylated histone H3 (Ac-Lys18,
07–354, Sigma), PARP-1 (ab227244, Abcam), apoptosis Western
blot cocktail (136812, Abcam), cleaved PARP-1 (ab32561, Abcam), and
HSC70 (sc-7298, Santa Cruz). Following overnight incubation, horseradish
peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody
(7076, Cell Signaling) or HRP-linked anti-rabbit IgG secondary antibody
(7074, Cell Signaling) was applied to the membrane in a 1:5000 dilution.
The bands were visualized using clarity Western ECL substrate luminal/enhancer
solution and peroxide solution. Western blotting analysis was carried
out using Image lab software (Bio-Rad).^{47 (link),50 (link),70 (link)}