Anti tau

The primary antibodies used in this study were anti-actin (Sigma, AC-40), anti-BiP (Cell Signaling Technology, 3177), anti-CHOP (Cell Signaling Technology, 2896), anti-p-eIF2α (Cell Signaling Technology, 3398), anti-eIF2α (Cell Signaling Technology, 9722), anti-tau (Dako, A0024), anti-GFAP (Dako, Z0334), anti-NeuN (Cell Signaling Technology, 12943), anti-GAPDH (Abcam, ab8245), and MC-1 and PHF-1 (gifts from Prof. Peter Davies) (17 (link)). Secondary antibodies were conjugated to Alexa Fluor fluorophores (Invitrogen) or IRDyes (LI-COR Biosciences). The plasmids pRK5-EGFP-TauP301L and pRK5-EGFP-Tau were gifts from Karen Ashe (Addgene plasmids 46908 and 46904, respectively) (32 (link)), and GFP in the constructs was exchanged for RFP.
Primers were obtained from Eurofins Genomics: BiP, 5-ATTGGAGGTGGGCAAACCAA-3 (forward) and 5-TCGCTGGGCATCATTGAAGT-3 (reverse); CHOP, 5-TCCCCAGGAAACGAAGAGGAAG-3 (forward) and 5-TCATGCGTTGCTTCCCAGGC-3 (reverse); XBP-1, 5-GGAGTGGAGTAAGGCTGGTG-3 (forward) and 5-GTCCAGAATGCCCAAAAGGATA-3 (reverse). Primers for the reference gene eIF4a were purchased from Primer Design.

20 fly heads were homogenized in 50 μl LDS sample buffer (Invitrogen) including 5% beta-mercaptoethanol (Sigma-Aldrich). Samples were boiled at 95 °C for 5 min and centrifuged at 15,000 g for 5 min at 4 °C. The supernatant was collected and stored at − 20 °C until loaded on 4–12% Bis-Tris NuPAGE gels (Invitrogen) using 5 μl of sample per well. Gel electrophoresis was performed at 140 V for 70 min and the gels were blotted on a PVDF membrane using XCell II (Invitrogen) at 30 V for 1 h. Membranes were blocked in 3% bovine serum albumin in tris buffered saline with 0.1% Tween20 (TBST) for 30 min and incubated with primary antibodies in blocking buffer over-night at 4 °C. Following washes in TBST, membranes were incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch) at 1:10,000 for 2 h, washed and the luminescent signal was developed using ECL prime (Amersham) and detected with Amersham Imager 600. Primary antibodies: anti β-tubulin (CAT#E7, DSHB, 1:500), anti β-Galactosidase (CAT#Z378A, Promega, 1:2000), anti-TDP-43 (CAT#10782, Proteintech, 1:1000), anti Tau (CAT#A0024, Dako, 1:1000).