Polyclonal anti myc

Cultured cells were fixed in 4% paraformaldehyde (PFA) for 10 min, rinsed 3X in PBS, and permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 10 min. Cells were then blocked in 5% milk for 1 hr and subsequently incubated with primary antibodies of interest overnight at 4°C following standard immunofluorescence techniques. Cell-based nuclear stippling and cytoplasmic aggregation of lysine mutants was performed by immunofluorescence microscopy (40X magnification) using Olympus BX 51 microscope. Stippling and aggregation phenotypes were quantified as a percent of transfected cells using >10 fields and sampling error was calculated using the standard error of the mean (SEM). Statistical analysis was determined using a two-tailed unpaired t-test with unequal variance (significance set at p-values < .05). All quantitative fluorescence was independently validated with a minimum of N=3 biological replicates. Primary antibodies used for immunofluorescence were as follows: polyclonal anti-TDP-43 (Proteintech, 1:500), polyclonal anti-myc (1:4000, Sigma), monoclonal anti-myc 9E10 (Sigma, 1:1000), monoclonal anti-Hsp70 (Stressgen, 1:500).