Hspa4

For analysis of protein levels by Western blot, whole tissue lysates were used from either the proteomic analysis or were prepared as described previously (34 (link)). Proteins were separated on precast SDS-PAGE 4–12% criterion gels (Bio-Rad) and transferred to polyvinylidene difluoride or nitrocellulose membranes. Site-specific antibodies directed to acetylated α-tubulin (Sigma, T7451), HSPA1 (Enzo Life Sciences, ADI-SPA-810), HSPA2 (Proteintech group, 66291-1), HSPB1 (Enzo Life Sciences, ADI-SPA-800), HSPB5 (Enzo Life Sciences, ADI-SPA-223), HSPB7 (abcam, ab150390), HSPD1 (Enzo Life Sciences, ADI-SPA-805), HSPA4 (Cell Signaling, 3303S), HSP90 (Cell Signaling, 4874S), α-tubulin (Sigma, T9026), tyrosinated tubulin (Sigma, T9028), detyrosinated tubulin (abcam, ab48389), and GAPDH (Cell Signaling, 2118S; Fitzgerald, 10R-G109a) were used to detect the proteins which were visualized with an enhanced chemiluminescence detection kit (Amersham) and scanned with Amersham Imager 600. Protein levels were determined by densitometric analysis and normalized to GAPDH.

Samples in soluble fraction were loaded into a 4–20% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad), and transferred to PVDF Immobilon FL membranes (Millipore). After blocking with 5% non-fat milk in PBS, membranes were blotted overnight with primary antibodies in 5% non-fat milk containing 0.01% Tween-20, and then probed with LI-COR IRDye secondary antibodies or horseradish peroxidase-conjugated secondary antibody, detected by SuperSignal West Femto Chemiluminescent Substrate (Pierce). The information of primary antibodies and their dilutions used in this study are as follows: Cleaved Caspase-3 (Cell Signaling Technology, 9661, 1:1000), Caspase-3 (Cell Signaling Technology, 9662, 1:1000), PSD95 (Abcam, ab2723, 1:1000), Synaptophysin (Abcam, ab8049, 1:1000), AT8 (Thermo Fisher Scientific, MN1020, 1:1000), PHF1 (Abcam, ab184951, 1:1000), Tau5 (Millipore, 577801, 1:1000), APP (Thermo Fisher Scientific, 14-9749-82, 1:1000), GFAP (Millipore, MAB360, 1:1000), EEA1 (Cell Signaling Technology, 2411, 1:1000), LAMP1 (Cell Signaling Technology, 9091, 1:1000), G3BP (BD, 611126, 1:1000), ERCC4 (Fitzgerald, 10R-4026, 1:1000), POLR3A (Abcam, ab96328, 1:1000), and HSPA4 (Cell Signaling Technology, 3303, 1:1000). All uncropped blot images with molecular size markers were available in the Source data file.