Western Blot Analysis of Hippocampal Proteins

Western blot analysis was performed as previously described^{9 (link)}. Briefly, dorsal hippocampal samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (HybondP, GE Healthcare UK Ltd.). The membrane was blocked with a blocking agent (GE Healthcare) and then incubated at 4 °C overnight with the following primary and secondary antibodies: mouse monoclonal anti-MAOB (1:1,000; Santa Cruz Biotechnology, Inc., sc-515354), mouse monoclonal anti- TIEG2 (KLF11) (1:1,000; Santa Cruz Biotechnology, sc-136101), rabbit polyclonal anti-p-HP1γ (Ser83) (1:5,000; Invitrogen, Thermo Scientific, PA517210), mouse monoclonal anti-HP1γ (1:5,000; Santa Cruz Biotechnology, sc-398562), mouse monoclonal anti-actin (1:10,000; Santa Cruz Biotechnology, sc-8432), horseradish peroxidase-conjugated secondary antibody against mouse or rabbit (1:20,000; Santa Cruz Biotechnology, sc-2357 for mouse, sc-2004 for rabbit). The antibody-reactive bands were visualized using a chemiluminescent substrate kit (GE Healthcare). Bands were analyzed by densitometry, using ImageJ (https://imagej.nih.gov/ij/).

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Hashikawa-Hobara N., Fujiwara K, & Hashikawa N. (2024). CGRP causes anxiety via HP1γ–KLF11–MAOB pathway and dopamine in the dorsal hippocampus. Communications Biology, 7, 322.

Publication 2024

HybondP blocking agent mouse monoclonal anti-actin horseradish peroxidase-conjugated secondary antibody chemiluminescent substrate kit

Corresponding Organization : Okayama University of Science

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein levels of MAOB
Protein levels of TIEG2 (KLF11)
Phosphorylation levels of HP1γ (Ser83)
Protein levels of HP1γ
Protein levels of actin

control variables

Dorsal hippocampal samples
SDS-PAGE for protein separation
Polyvinylidene difluoride membranes for protein transfer
Blocking agent used for membrane blocking
Primary and secondary antibodies used for protein detection

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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