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. Author manuscript; available in PMC: 2016 Aug 3.
Published in final edited form as: FEBS J. 2015 Mar 27;282(10):2045–2059. doi: 10.1111/febs.13259

Fig. 1.

Fig. 1

15d-PGJ2 binds to PDI in vitro and in primary neurons. (A) PDI recombinant protein was preincubated with or without 500 μM 15d-PGJ2 for 30 min before being incubated with vehicle or 5 μM b-15d-PGJ2 for another 90 min. The b-15d-PGJ2–PDI adducts were detected by immunoblotting with streptavidin–HRP. (B) 15d-PGJ2 binds to PDI in primary neurons. (Upper) Rat primary neurons were incubated with 10 μM 15d-PGJ2 or b-15d-PGJ2 for 2 h prior to harvest. The avidin pull-down assay was performed with cell lysates, and b-15d-PGJ2–PDI adducts were detected with PDI antibody. (Lower) Rat primary neurons were incubated with 10 μM b-15d-PGJ2 (+) or vehicle (−) for 2 h before harvest. Cell lysates were either subjected to immunoblotting to detect biotinylated proteins with streptavidin–HRP (Str-H, upper left) and PDI levels with an PDI antibody (lower left) or subjected to IP to detect the b-15d-PGJ2–PDI adduct (right). For IP, cell lysates were incubated with PDI antibody-conjugated resin (R) overnight before elution. A nonreactive control resin was included as a negative control. The b-15d-PGJ2–PDI adduct and PDI in the eluent were detected by immunoblotting with streptavidin–HRP (upper right) and PDI antibody (lower right), respectively. The arrow indicates the band representing b-15d-PGJ2–PDI adduct. (C) Avidin pull-down assay detecting arachidonic acid (AA) metabolite-modified PDI in primary neurons. Neurons were incubated with b-arachidonic acid (b-AA) then underwent hypoxia (+) or normoxia (−) before being harvested at the indicated time points. (Upper) Avidin-bead-bound PDI was detected by immunoblotting with PDI antibody. (Lower) Biotinylated proteins and endogenous PDI in cell lysates were detected by immunoblot with streptavidin–HRP and PDI antibody. (D) Fragmentation spectra of 15d-PGJ2-modified PDI (Uniprot Accession: P07237). The tryptic peptides indicate cysteine (C*) modifications at C-57 or C-60 (upper) and C-401 or C-404 (lower). Cysteine carbamidomethylation from sample processing for mass spectrometry is denoted by C#. MS/MS has an abundance of y- and b-ions N terminal to proline (underlined) with mass shifts corresponding to 316.204 Da indicating 15d-PGJ2 adduction.