A–C. Trx80 immunostaining in (A) human brain, (B) human mixed primary culture of neurons and astrocytes, (C) pure neuronal human primary culture. Trx80 is seen in neurons, particularly in bipolar and pyramidal neurons (right panel in A). Arrowheads in B indicate astrocytes. The pure neuronal cultures show Trx80 staining in soma and neuronal processes (C). Scale bars: 25 µm.
D. Trx80 immunoblotting of samples from three control brains. The 7D11 antibody recognizes a band at approximately 30 kDa.
E. Trx80 inmunoblotting of lysates from different cell types (1: Hela, 2: U2020, 3: U937, 4: SH-SY5Y, 5: SH-SY5Y overexpressing Trx1, 6: human primary neurons, 7–8: human primary glia).
F. Trx80 is secreted and detected in the media from rat neuronal primary cultures and SH-SY5Y cells.
G. Immunoblotting with the anti-Trx80 antibody 4H9. Recombinant Trx80 was incubated O/N at 37°C and compared to fresh Trx80. Samples were mixed with sample buffer ± DTT (10 mM) and left untreated or heated to 95°C.
H. Transfected Trx80 also migrates as a band of approximately 30 kDa, as the endogenous Trx80. Panel shows two different clones.
I. Reduction of Trx1 expression by siRNA resulted in decreased levels of the 30 kDa band detected by 7D11. Scrambled RNA (scRNA)-treated and vehicle-treated cells were used as controls.
J. Lysates from SH-SY5Y cells were incubated (37°C, 24 h) with or without 2.4 µg of recombinant Trx80, Trx1 or both peptides together. As controls, Trx80 and Trx1 peptides were incubated at 37°C for 24 h in PBS. Blotting with anti-Trx80 (7D11) and anti-Trx1 abs is shown, respectively, in the left and right panels. Incubation of recombinant Trx80 with cell lysates resulted in more aggregated signals (left panel, lanes 2, 4 and 6). Addition of recombinant Trx1 alone or in combination with Trx80 did not change the aggregation pattern of Trx80 (left panel, lanes 3 vs. 2; 4 vs. 5; and 6 vs.7). The aggregation pattern of Trx1 was also changed by incubation with lysates, with the appearance of more dimers and trimers (right panel, lane 1 vs. 5). Addition of Trx80 did not change the Trx1 aggregation pattern (right panel, lanes 1 vs. 3 and 5 vs. 7).
