Mouse anti hnrnp a1

Chicken anti-GFP (1:1000, Abcam), Rabbit anti-GFP (1:5000, Invitrogen), mouse anti-hnRNP-A1 (1:1000, Millipore, clone 9H10), Rabbit anti-Nucleolin (1:2000, Abcam, ab22758), Rabbit anti-HDAC4 (1:1000, Abcam, 12172), Mouse anti-TDP43 (1:1000, Abcam, 57105), Mouse anti-γH2AX-pS139 (1:1000, Abcam, 26350), Mouse anti-FLAG (1:2000, Sigma, F3165), Rabbit anti-AAV VP1, VP2, VP3 (1:1000 (WB), 1:50 (ICC), #03-61084, American Res Prod, Waltham, MA), Mouse anti-Integrin αVβ6 (Millipore, MAB2077Z) and mouse anti-NeuN (1:300, Millipore) were used at the dilutions listed above. Alexa Fluor dye (546, 660-C2) maleimide dye (Life Techologies) and Biotin-maleimide (B1267, Sigma) was maintained in aliquots at a stock concentration of 10 mM in DMSO at −20 °C. LC/MS grade acetonitrile (Pierce), ammonium bicarbonate (Sigma), and Pierce™ Trypsin Protease, MS Grade (90057), and formic acid (Sigma) were used for LC/MS described below.

Neuro-2a cells were plated onto poly-D-lysine (Sigma-Aldrich) coated six-well plates and transfected as described above and harvested for Western blotting at 72 h after siRNA transfection. Neuro-2a cells were lysed in CytoBuster (Millipore) protease extraction reagent containing protease inhibitors (Roche) as per the manufacturer’s protocol. Cell lysates were separated by SDS-PAGE before being transferred to PVDF membrane. Membranes were blocked in 10% normal goat serum for 1 h at room temperature before being placed into primary antibody for overnight incubation at 4°C. The following primary antibodies were used: mouse anti-β-actin (1:1000; Cell Signaling Technology, RRID: AB_2242334) and mouse anti-hnRNP A1 (1:1000; Millipore, RRID: AB_10562650). Membranes were washed and incubated with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3000; Bio-Rad, RRID: AB_11125547). Membranes were developed using Clarity Western ECL substrate (Bio-Rad) and visualized using the Bio-Rad ChemiDoc system. Three biological replicates of siNEG and siA1 transfected Neuro-2a cells were harvested and run for Western blotting. Blots were analyzed in ImageJ (RRID: SCR_003070) by densitometry and normalized to β-actin signal.

Tissue was sectioned at 5 µm and mounted onto slides before undergoing deparaffinization and rehydration through a xylene and ethanol gradient. Endogenous peroxidases were blocked using 0.2% hydrogen peroxide in methanol. Heat‐mediated antigen retrieval was performed by placing slides in 10 mmol/L Tris/Ethylenediamine tetraacetic acid (EDTA) buffer for 45 min in a steamer. Sections were blocked in 10% fetal bovine serum (FBS) in phosphate buffered saline (PBS) for 15 min before being incubated with primary antibodies diluted in the same solution overnight at 4°C. The following primary antibodies were used: rabbit anti‐TDP‐43 (Novus Biologicals, Centennial, CO) and mouse anti‐hnRNP A1 (Millipore, Burlington, MA). The next day, sections were washed and incubated with the appropriate biotinylated secondary antibody for 1 h at room temperature and then incubated in avidin peroxidase for 1 h at room temperature. Slides were developed using 3,3′‐diaminobenzidine and counterstained with hematoxylin.