Ab84078

The following commercial primary antibodies were used: FUS full protein (rabbit polyclonal, 11,570–1-AP); FUS N-terminus (rabbit polyclonal, Abcam, ab84078; aa. 1–50); FUS C-terminus (Bethyl, A300-294A; aa. 500–526); p54nrb/NONO (rabbit polyclonal C-terminal, Sigma); SFPQ (rabbit monoclonal, ab177149, Abcam; rabbit polyclonal, A301-322A, Bethyl); beta-actin (mouse monoclonal, A5441, Sigma). Antibodies were used at 1:500–1:1000 dilution for all applications unless stated otherwise.

The protein extraction protocol from cancer cells was previously reported [30 (link)]. Briefly, cells were collected and lysed using Radio Immunoprecipitation Assay (RIPA) protein extraction reagent (Beyotime) with a protease inhibitor cocktail (Roche, IN, USA). Equal amounts of protein lysate were quantified using BCA kit (Thermo Fisher, USA), separated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then sealed with 5% non-fat milk for 1 hour, and incubated with primary antibodies (Abcam, Cambridge, UK) against: FUS (ab84078, 1/1000), SLC10A1 (ab131084, 1/1000), β-actin (ab8227, 1/1000), GLUT1 (ab14683, 1/2500), HK2 (ab227198, 1/5000), LDHA (ab47010, 1/1000), PGK1 (ab154613, 1/1000) overnight at 4°C, with GAPDH (ab9485, 1/2500) as control. After washing, the membrane was incubated with goat anti-rabbit secondary antibody (ab96899, 1/1000, Abcam, Cambridge, UK) at 37°C for 1 hour. The protein bands were finally detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) according to manufacturer's instructions.

Paraffin sections (5 µm) were mounted on poly-L-lysine coated slides and placed to dry (37 °C overnight) and then processed for immunohistochemistry described in detail elsewhere [75 (link), 76 (link)]. At first, the sections were deparaffinized in xylene for 20 min and then rehydrated in 100%, 96%, and 70% ethanol for 5 min each followed by endogenous peroxidase quenching (0.3% H₂O₂ in methanol) for 20 min. Antigen retrieval was performed in these sections by heating them in citrate buffer, pH 6 (DAKO), for 20 min in a pressure cooker. After washing in PBS, sections were incubated with primary PML antibody (EPR16792, ab179466, 1:100) and C90RF72/C9RANT poly-GA antibody (MABN889, Millipore, 1:100), FUS antibody (ab84078, Abcam, 1:100) or phosphorylated TDP-43 (pTDP-43) antibody (TIP-PTD-M01, Cosmo Bio Co.LTD, 1:100) for 1 h at room temperature or 4 °C overnight. After washing in PBS, sections were incubated with appropriateHRP-linker secondary antibody (ImmunoLogic, Duiven, The Netherlands) for 30 min at room temperature. DAB reagent (ImmunoLogic, ready to use) was used to visualize antibody binding. The sections were then counter-stained with 6% hematoxylin for 3 min. All procedures were performed at room temperature.