Anti pfn1

As described previously (Fil et al., 2017 (link)), transgenic (hPFN1^WT and hPFN1^G118V) mice were generated, and the selected mice with and without ALS-like symptoms were genotyped to determine the presence of the transgene. All the animal work was approved by UAMS IACUC (University of Arkansas for Medical Sciences, Institutional Animal Care and Use Committees) and carried out accordingly. Tissues used in this study were the tissue samples from mutant and wild-type PFN1 mice that were previously isolated as part of the research described in Fil et al. (2017) (link) and were stored for further use. To analyze the expression level of hPFN1 in the tissues from these mice, brain, spinal cord, liver, and leg muscles were isolated and stored at −80°C till use. Then the frozen tissues were thawed out on the ice, and the whole brain, spinal cord, liver, and leg muscles were homogenized with RIPA buffer. After estimating protein concentration, an aliquot was mixed with sample buffer (Invitrogen), heated at 90°C for 10 min, and run on 4%–12% Bis-Tris Gel (Invitrogen). Then the separated proteins were analyzed by western blotting. Anti-PFN1 and anti-β-actin (Sigma Aldrich) were the primary antibodies followed by appropriate secondary antibodies.