Anti rat

First Probing: HS (1:1,000, 10E4, cat no H1890, US Biological, MA, USA), Syn-1 (1:750, 281-1, cat no 553712, BD Pharmingen, Brøndby, Denmark), Syn-4 (1:750, KY8/2, cat no 550350, BD Pharmingen), CD44 (1:200, DAKO, Glostrup, Denmark). Second probing: CS (1:1,000, CS-56, cat no C8035, Sigma-Aldrich, Brøndby, Denmark), syndecan-3 (1:1,000, cat no AF3539, R&D Systems, UK). HRP-conjugated secondary antibodies: anti-rabbit (1:2,000), anti-rat (1:4,000), and anti-mouse (1:3,000) (DAKO, Glostrup, Denmark). All syndecan antibodies detect the syndecan protein structure and not carbohydrates.

Whole-cell extracts were resuspended in PBS in the presence of protease inhibitor and sonicated. 15 μg of protein sample were separated on  a 10% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (Invitrogen). The membrane was blocked in 4% milk-PBST (PBS with 0.1% Tween-20) and proteins were stained using the following antibodies: anti-TDP-43 (rabbit, Proteintech, 1:1000), anti-GAPDH (rabbit, Proteintech, 1:1000), anti-HSP70 (rat, EnzoLife Science, 1:1000), anti-tubulin (mouse, available in house, 1:10,000) and HRP-conjugated secondary antibodies anti-rabbit (goat, Dako, 1:2000), anti-mouse (goat, Dako, 1:2000), anti-rat (rabbit, Dako, 1:2000).
To detect TBC1D1, 30 μg of protein sample were separated using a pre-cast gradient gel (4–12%, Invitrogen) and wet blotted onto a PVDF membrane (Merck Millipore). The membrane was then blocked in 4% milk-TBST, stained with anti-TBC1D1 (rabbit, Cell Signalling, 1:1 000, in 2% milk-TBST) and anti-rabbit HRP-conjugated secondary antibody (goat, Dako, 1:2 000).
Image acquisition and result quantification were conducted using Alliance Q9 Advanced Chemiluminescence Imager (UviTech).