Western Blotting of Neural Proteins

Western blotting was completed as previously described [27 (link)]. Whole cell lysates were generated using RIPA buffer with protease and phosphatase inhibitors (Sigma and ThermoFisher). Briefly, equal amount of protein were resolved via SDS-PAGE on Criterion TGX gels 4–15% (BioRad). Gels were transfered to PVDF membranes and blocked with 5% BSA in PBST. Primary antibodies were incubated overnight at 4°C followed by three washes with PBST. Secondary antibodies (ThermoFisher) were incubated at room temperature for one hour. Gels were imaged using WestFemto ECL (ThermoFisher) and a BioRad ChemiDoc XRS imaging system. Loading controls included either actin (Cell Signaling) or AmidoBlack (Sigma) total protein stain. Primary antibodies include AβPP 6E10 (Biolegend), BACE1 (Abcam), COX41I (Cell Signaling), Calreticulin (Cell Signaling), GAPDH (Cell Signaling), GFAP (Abcam), HDAC (Cell Signaling), MAP2 (Abcam), Nestin (ThermoFisher), S100β (Abcam), and Synaptophysin (Abcam).

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Wilkins H.M., Troutwine B.R., Menta B.W., Manley S.J., Strope T.A., Lysaker C.R, & Swerdlow R.H. (2022). Mitochondrial Membrane Potential Influences Amyloid Precursor Protein Localization and Aβ Secretion. Journal of Alzheimer's disease : JAD, 85(1), 381-394.

Publication 2022

protease and phosphatase inhibitors Criterion TGX gels 4–15% WestFemto ECL ChemiDoc XRS imaging system actin AmidoBlack BACE1 Calreticulin GAPDH GFAP Nestin Synaptophysin

Actin Antibodies Bace1 Buffer Calreticulin Cell Gapdh Gels Gfap Inhibitors Map2 Membranes Nestin Phosphatase Protease Protein Pvdf Ripa S100β Sds page Stain Synaptophysin

Corresponding Organization : University of Kansas

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of AβPP, BACE1, COX41I, Calreticulin, GAPDH, GFAP, HDAC, MAP2, Nestin, S100β, and Synaptophysin

control variables

Protein amounts loaded were equal
Actin or AmidoBlack total protein stain used as loading controls

controls

Positive controls: None explicitly mentioned
Negative controls: None explicitly mentioned

Annotations

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