Anti tubulin t9026

Western blotting was performed as described previously (35 (link), 36 (link)). Ten fly heads for each genotype were homogenized in Tris-Tricine SDS sample buffer (Novex, ThermoFisher) and the same amount of the lysate was loaded to each lane of 10–20% Tris-Tricine gels (Novex, ThermoFisher). Separated proteins were transferred to nitrocellulose membrane and blotted with anti-Aβ (6E10, Biolegend) or anti-tubulin (T9026, Sigma-Aldrich) antibodies. After incubating with secondary antibody, the membranes were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) and analysed by ImageQuant LAS 4000 (GE Healthcare Life Sciences). The signal intensities were quantified using Image J (NIH).

Reagents and antibodies were purchased from the following suppliers: HRP–conjugated goat anti-mouse IgG (31430) and goat anti-rabbit IgG (31461); Alex Fluor 488 goat anti-mouse IgG (A11029), Texas Red goat anti-mouse IgG (T-862), and goat anti-rabbit IgG (T-2767); and Alexa Fluor Plus 647 goat anti-mouse IgG (A32728) and goat anti-rabbit IgG (A32733) are from Thermo Fisher Scientific. Anti-beclin-1 (NB500-249), anti-FAM134B (NBP2-55248), and anti-Sec62 (NBP1-84045) are from Novus Biologicals. Anti-calnexin (C4731), anti-HA (H9658), anti-LC3B (L7543), anti-Myc (C3956), and anti-tubulin (T-9026) are from Sigma-Aldrich. Anti-ERGIC53 (ALX-804-602) is from Enzo Life Sciences. Anti-GABARAP+L1+L2 (ab109364) and anti-GM130 (ab52649) are from Abcam. Anti-LAMP2 (555803) and anti-Sec31A (612351) are from BD Pharmingen. Anti-p62 (H00008878-M0162) is from Abnova. Anti-VMP1 (12978s) is from Cell Signaling. DC Protein Assay (500-0119) and Clarity Western ECL Substrate (170-5061) are from Bio-Rad. Effectene Transfection Reagent (301427) is from Qiagen. Lipofectamine RNAiMAX Reagent (100014472) is from Thermo Fisher Scientific. MTS cross-linking reagents M3M (1,3-propanediyl bis-MTS) and M8M (1,5-pentanediyl bis-MTS) are from Toronto Research Chemicals. ProLong Diamond antifade mountant without (P36970) or with DAPI (P36971) is from Thermo Fisher Scientific.

Whole cell extracts from siRNA treated samples was collected and resolved by SDS-PAGE. Samples were transferred to a PVDF membrane at 150 mA for 4 h, blocked with 5% milk in TBST (10 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20) for 1 h and incubated in primary antibody—1:1000 anti-CHD4 #39289 (Active Motif), 1:10 000 anti-tubulin T9026 (Sigma), 1:500 anti-CHD3 (ab109195), 1:10 000 anti-PARP1 (rabbit polyclonal, (5 (link))), 1:1000 anti-Alc1 (affinity purified rabbit polyclonal, (5 (link)))—in 1% milk in TBST overnight. Primary antibodies were detected using anti-mouse or anti-rabbit HRP-conjugated IgG (BioRad) diluted 1:10 000 in 1% milk in TBST. The blots were developed using WesternBright ECL-spray (Advansta).