Protein Extraction and Western Blot Analysis

Total protein was extracted in RIPA buffer supplemented with 10% proteinase inhibitor cocktail (Merck) and quantified using Pierce BCA protein assay kit (ThermoFisher Scientific). For KAT2B western, NE-PER Nuclear and Cytoplasmic extraction reagents (ThermoFisher Scientific) were used to enrich for nuclear proteins. PVDF membranes were incubated overnight with 1/1000 Cas9 (#14697, Cell Signalling)^{18 (link)}, 1/2000 B-Actin (#ab8227, Abcam)^{19 (link)}, 1/10,000 GAPDH (Abcam, ab128915)^{20 (link)}, 1/100 KAT2B (#sc-13124, Santa Cruz Biotechnology)^{21 (link)}, followed by 1 h incubation with HRP-conjugated secondary antibodies. Membranes were developed using SuperSignal West Pico PLUS Chemiluminescent substrate (ThermoFisher Scientific) and bands detected using iBright CL1000 (Invitrogen).

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Pavlou S., Foskolou S., Patikas N., Field S.F., Papachristou E.K., Santos C.D., Edwards A.R., Kishore K., Ansari R., Rajan S.S., Fernandes H.J, & Metzakopian E. (2023). CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death. Scientific Reports, 13, 3934.

Publication 2023

proteinase inhibitor cocktail Pierce BCA protein assay kit NE-PER Nuclear and Cytoplasmic extraction reagents B-Actin GAPDH KAT2B SuperSignal West Pico PLUS Chemiluminescent substrate iBright CL1000

Actin Antibodies Assay Buffer Cytoplasmic Gapdh Membranes Nuclear proteins Protein Proteinase inhibitor Pvdf Ripa

Corresponding Organization : UK Dementia Research Institute

Other organizations : Cancer Research UK

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

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control variables

Total protein was extracted in RIPA buffer supplemented with 10% proteinase inhibitor cocktail (Merck)
PVDF membranes were incubated overnight with 1/1000 Cas9 (#14697, Cell Signalling), 1/2000 B-Actin (#ab8227, Abcam), 1/10,000 GAPDH (Abcam, ab128915), 1/100 KAT2B (#sc-13124, Santa Cruz Biotechnology)

positive controls

B-Actin (#ab8227, Abcam)
GAPDH (Abcam, ab128915)

negative controls

Not explicitly mentioned

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