Fig 1: Quantitative analysis of proteomics after injury identifies downregulation of Kif5a synthesis and transport after injury.(a) Immunoprecipitation (IP) of biotinylated protein from retinal and optic nerve samples, probed for Sncb, Gap43, or Arf3. Input of each sample on top row, subsequent IP of each sample on bottom row, for either optic nerve crush (ONC) samples or sham surgeries. (b) Volcano plot comparing biotinylated proteins from control versus ONC nerve samples. Normalized spectral abundance factor (NSAF) values for each sample type were averaged across three replicates. Proteins with an absolute fold change greater than 1.5, p-value less than 0.05, or both, are colored blue, gray, and red respectively. (c) Retinal cross-sections immunostained with Kif5a and DAPI showing localization of Kif5a in the retinal ganglion cell (RGC) layer (top) and RGC-specific marker RBPMS and Kif5a showing co-localization of Kif5a with RGCs (bottom). Scale bar, 100 µm. (d) Three replicates of biotin IPs probed with an antibody against Kif5a. Inputs are on the left. The IP was stripped and re-probed for biotin for a measurement of total biotinylated protein pulled down (not shown). (e) Quantification of the change in transported Kif5a compared to the change in total transported protein after ONC. One-sample, two-tailed t-test, p = 0.003, n = 3. The bar height and error bar represent the mean and 95% confidence interval respectively. ONC = optic nerve crush. (f) Average intraocular pressure after induction of glaucoma over time in two groups, one with sustained pressure elevation and one with only initial pressure elevation. (g) Quantitative changes in retinal protein synthesis 3 weeks after induction of glaucoma. 1143 and 1457 proteins were quantified in replicates 1 and 2, respectively. Log2 transformed normalized spectral ratios of glaucoma/control samples were plotted from lowest to highest. Newly synthesized Kif5a, detected by BONCAT and quantitative mass spectrometry, was decreased in both replicates, while Kif5b synthesis was unaffected. Figure 2—source data 1.Raw western blots for Figure 2.
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