Ab32561

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)}

Immunohistochemistry of the Drosophila adult brain was performed as previously described [44] (link). To stain ORN cell bodies, we dissected antennae from flies, fixed them in 4% (vol/vol) paraformaldehyde/0.3% (vol/vol) phosphate-buffered saline with Triton X-100 (PBT) at room temperature (R.T.) for 30 min, mounted them in OCT, and cut 14 µm-thick sections on a cryostat. Slides were then re-fixed with 4% (vol/vol) paraformaldehyde/0.3% (vol/vol) PBT at R.T. for 30 min, washed with 0.3% (vol/vol) PBT, and labeled using standard techniques. Antibodies used include rat anti-mouse CD8 antibody (1∶100, MCD0800, Invitrogen), rabbit anti-cleaved PARP (Asp214) antibody (1∶100, #9541, Lot.7, Cell Signaling), rabbit anti-cleaved PARP antibody [Y34] (1∶100, ab32561, Abcam), nc82 mouse monoclonal antibody (1∶40, Developmental Studies Hybridoma Bank), anti-rat Alexa488 (1∶250), anti-rabbit Cy3 (1∶1000), and anti-mouse Cy5 (1∶1000) (Jackson Laboratory). Confocal images were captured using a Leica SP5 confocal microscope.

To measure the level of cleaved PARP, cells were fixed with 75% (v/v) ethanol in PBS, permeabilized with 0.25% (v/v) Tween 20 in PBS and stained overnight with a rabbit antiserum specific for cleaved PARP (Rabbit anti PARP cleaved (Y34), #ab32561, Abcam).

Ice-cold RIPA buffer (Cell Signaling, USA) was used to lyse the cells. Total protein lysates (50 μg) were separated by denaturing 10% SDS-PAGE and electrotransfered onto a nitrocellulose membrane (Millipore, USA) for subsequent blotting with primary antibodies. The primary antibodies were: IMP4 (1 : 1000, ab181046; Abcam), Bcl-2 (1 : 1000, #15071; CST), Bax (1 : 1000, ab32503; Abcam), PARP (1 : 1000, ab191217; Abcam), E-cadherin (1 : 1000, 20874-1-AP; Proteintech, Wuhan), N-cadherin (1 : 1000, ab280375; Abcam), Vimentin (1 : 1000, ab20346; Abcam), glucose transporter 1 (GLUT1; 1 : 1000, ab115730; Abcam), hexokinase II (HK2; 1 : 1000, ab209847; Abcam), cleaved fructose phosphate kinase P (PFKP; 1 : 1000, ab32561, Abcam), pyruvate kinase M2 (PKM2; 1 : 1000, ab85555; Abcam), lactate dehydrogenase A (LDHA; 1 : 1000, #3582; Cell Signaling, USA), MEK1 (1 : 1000, ab32091; Abcam), p-MEK1 (1 : 1000, ab96379; Abcam), ERK (1 : 1000, ab184699; Abcam), p-ERK (1 : 1000, ab201015; Abcam), p21 (1 : 1000, ab109520; Abcam), p53 (1 : 1000, ab75754; Abcam), cyclin D1 (1 : 1000, ab16663; Abcam), and GAPDH (1 : 1000, ab181603; Abcam), according to the manufacturers' protocols. After incubation with secondary antibodies for 60 min, protein bands were visualised using ECL kit (Beyotime).

Cytoplasmic protein and nuclear protein were extracted using a Nuclear and Cytoplasmic Extraction Reagents kit (78835, Thermo Fisher Scientific, USA). The protein concentration was measured by the BCA Assay Kit (23223, Thermo Scientific, USA). The protein was electrophoresed with 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and subsequently transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Specific primary antibodies (ab181602, ab32535, ab39012, ab41037, ab188570, ab186414, ab32042, ab32561, ab219413, ab76429, ab133462, ab32536, ab32511, Abcam, Cambridge, UK) (PA5-61136, Invitrogen, USA) were co-incubated with the membrane overnight at 4 °C. The membranes were rewarmed for 2 h, and then incubated with anti-rabbit IgG (ab97051, Abcam, Cambridge, UK) at 37 °C for 2 h. ECL Western blot kit (32209, Thermo Fisher Scientific, USA) was used to detect the bands. GAPDH and Lamin B were used as controls.